Lab Session One

Title: Preparation of Solutions

Objectives:

 Know different methods of expressing strength of solution

 Carry out different calculations.
Introduction: A solution is a homogenous mixture of two or more substances. These mixtures
can be in any form of matter. We talk about solutions in terms of concentrations, how much of
each substance, or solute, the solution contains. Concentration can be recorded and reported in
many different ways depending on the solution, example. Molarity – Moles/Liter; mols/L (M),
Mass/volume – grams/L; g/L or mg/ml, Normality – moles of active ions/L (N).

In a laboratory setting, solutions are an essential part of research. Most biochemical reactions
occur in liquid solutions. Buffers, reaction mixtures, cell culture media, cell lysates, liquid acids
and bases are all examples of solutions commonly used in the lab.

For making a solution; there is a need to know which is being dissolved, which is being used to
dissolve the solute, the desired concentration and the desired volume of the substance that you
want to prepare. In this sections we try to perform different diluting solutions preparation
techniques, using dilutions to make complex solutions and making a complex solution.

Steps in Solution Preparation

1. Refer to the laboratory manual for any specific instructions on preparation of the
particular solution and the bottle label for any specific precautions in handling the
chemical.
2. Weigh out the desired amount of chemical(s)/solutes.
3. Place chemical(s) into appropriate size beaker with a stir-bar.
4. Add less than the required amount of water. Prepare all solutions with DDH2O
5. When the chemical is dissolved, transfer to a graduated cylinder and add the required
amount of distilled water to achieve the final volume.
6. If the solution needs to be at a specific pH, check the pH meter with fresh buffer solutions

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 and follow instructions for using a pH meter.
7. Autoclave, if possible, at 121° C for 20 min. Some solutions cannot be autoclaved, for
example SDS. These should be filter sterilized through a 0.22 µm filter. Media for
bacterial cultures must autoclaved the same day it is prepared, preferably within an hour
or two. Store at room temperature and check for contamination prior to use by holding
the bottle at eye level and gents whirling it.

1. Simple Dilution (Dilution Factor Method), Mixing parts or volumes, Serial Dilution,
Percentage and Making fixed volumes of specific concentrations from liquid reagents

1. How to prepare 1:100 dilution of acid alcohol solution.

2. How to prepare 1:4 acid alcohol solution, phenol\chloroform (50:50)
3. How to prepare 1:10 serial dilution of soil/water sample for a bacterial culture.
4. How to prepare 37gm%w/w HCl, 10% w/v sodium dodecyl Sulphate (SDS), 20%(w/v)
SDS and 70% V/V ethanol.
5. How can you prepare 200 µl solution of tetracycline having a concentration of 50mg/ml
from 3ml stock solution with a concentration of 100mg/ml?
6. Prepare 50ml of 0.5M NaCl solution from 6M solution of NaCl.
7. Suppose you want to make 100ml of a 1X Triton-X solution using a 100X Triton-X stock
solution. How would you do it?

2. Using Dilutions to Make Complex Solutions

Make complex solutions from multiple liquid stock solutions by treating each dilution
individually and combine them together in one container and brings the volume up to your
interest.

Prepare 250ml of 0.1M NaCl and 0.5M Tris-HCl from a stock solution of 6M NaCl and 1M Tris-
HCl.

3. Making Molar and Normal solutions

1. Prepare 500ml of 5 M potassium acetate (Stored –20oC),

2. Prepare 200ml of 3 M potassium acetate
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 3. Prepare 500ml of 3 M sodium acetate (pH 5.2),
4. How to prepare 0.5M HCl solution.
5. Prepare 100ml of 0.2N NaOH solution.
6. Prepare 100ml of 1N HCl solution.

Note: See the MW on its package/ container. 20 mg/ml proteinase K, phenol\chloroform (50:50)

4. Making a Complex Solution

Solutions often have more than just one solute. To make a complex solution with two or more
solid solutes, treat each solute individually and add both/ all solutes to a container and bring to
volume with water or other solvent depending on your interest.

1. Prepare 100ml solution I

Tris base (25mM), EDTA (10mM), Glucose (50mM). Note: See the MW on its package/
container.

 Weigh the above salts and dissolve in 80ml of dd water and adjust the pH 8.0 using 1N
HCl. Make up the volume to 100ml. autoclave and store at 4˚C. Do not over autoclave
glucose will be charred.
2. Prepare 100ml solution II (prepare fresh each time)

NaOH------------ 0.2N
SDS-----------------1%
Prepare 0.2 N NaOH and store in a plastic reagent bottle. Prepare 1% SDS and autoclave.
Mix them in 1:1 ratio before use. Do not autoclave NaOH.

3. Prepare 100ml solution III

Prepare 100ml of 3 M potassium acetate pH 5.5. Adjust the pH with glacial acetic acid and
make up the volume to 100ml. autoclave and store at 4 oc. Or: 60ml 5M potassium acetate
(potassium acetate in 100ml H2O) + 11.5ml glacial acetate + 28.5ml H2O.

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 4. TKM 1 Buffer / Low salt buffer (500 ml) of distilled water

TrisHCl (10mM) pH 7.6, KCl (10 mM), MgCl 2 (10 mM) and EDTA (2mM)

5. TKM 2 Buffer / High salt buffer(100 ml) of distilled water

TrisHCl (10mM) pH 7.6, KCl (10 mM), MgCl 2(10 mM), EDTA (2mM) and NaCl (0.4 M)

6. TE Buffer 100ml of distilled water

TrisHCl (10mM) pH 8.0, EDTA (1mM)

7. CTAB Buffer A (EBA) Per 100 mL

 2% (w/v) hexadecyltrimethyl  1.4 M NaCl = 8.2 g

ammonium bromide (CTAB) = 2.0 g  4% (w/v) polyvinylpyrrolidone
 100 mM Tris (pH 8.0) (Use 1 M (PVP) = 4.0 g
stock) = 10 mL  0.1% (w/v) ascorbic acid =0.1 g
 20 mM EDTA (Use 0.5 M stock) = 1  10 mM β-mercapto ethanol (BME)*
mL (Use 14.3 M stock) =70 µL

8. Extraction Buffer B (EBB) Per 100 mL

 50 mM EDTA (Use 0.5 M stock) =2.5 mL  10 mM β-mercapto ethanol (BME)*

 100 mM NaCl = 0.6 g (Use 14.3 M stock) =70 µL

Lab session Two

Title: - Isolation of plasmid DNA

Objective: - To isolate plasmid DNA from E.coli bacterium source

Introduction

The success of DNA purification is dependent on the initial quality of the sample and its
preparation. It would be nice to have a simple, straightforward formula that applies to all
samples, but some specimens have inherent limitations. The integrity of purified DNA in

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solution could be compromised by nuclease, pH below 6.0 and above 9.0, heavy metals, UV
light, and oxidation by free radicals. EDTA is often added to chelate divalent cations required for
nuclease activity and to prevent heavy metal oxidative damage.

Procedures

1. Pick up a single colony from overnight grown culture of E.coli cells having plasmid and incubate it in to 10 ml
LB broth containing ampicillin (50ug/ml). Incubate the inoculated LB using shaker at 37 0c for overnight.
2. Pipette 1.5 ml culture in a 1.5 ml epp. tube
3. Spin for 8-10 min at 6,000rpm and discard the supernatant. Then place it on ice.
4. Resuspend cell pellet in 100μl of ice-cold solution I. Vortex gently, place on ice for 5 min and shift to RT.
 Solution I contains glucose/sucrose, Tris, and EDTA. Glucose/sucrose is added to
increase the osmotic pressure outside the cells. Tris used to maintain a constant buffer pH
(= 8.0). EDTA protects the DNA from degradative enzymes (called DNAses) by
binding/chelating divalent cations that are necessary for DNAse activity.
5. Thaw and add 200 μl of solution II at RT. Mix gently by inverting the tube five times (Do not
vortex).
 Solution II contains NaOH and SDS (a detergent). This alkaline mixture ruptures the
cells, and the detergent breaks apart the lipid membrane and solubilizes cellular proteins.
NaOH denatures both plasmid DNA and chromosomal DNA.
6. Add 150 µl of solution III. Mix gently by inverting the tubes. Place on ice for 5min.
 Solution III contains a mixture of acetic acid and potassium acetate. The acetic acid
neutralizes the pH, allow the DNA strands to renature. The potassium acetate also
precipitates SDS from solution, along with the cellular debris. Plasmid DNA renature and
remains in solution but E.coli chromosomal DNA, from a partially renature tangle at this
step, in which denatured proteins and SDS are also trapped. The whole complex gets
precipitated.
7. Spin for 10 min at 6-8000 rpm. This fractionation step separates the plasmid DNA from the
cellular debris and chromosomal DNA in the pellet.
8. Transfer immediately the supernatant to a fresh epp. tube and add 900 ul of solution IV. Mix
and keep at -20 oc for overnight.

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  Solution IV is Absolute alcohol/Isopropanol which effectively precipitates nucleic
acids, but it is much less effective with proteins. A quick precipitation can
therefore purify plasmid DNA from protein contaminants
9. Spin for 20 min at 10,000rpm for 30 min, at 6,000rpm. Decant the supernatant invert the vial
on blotting paper to drain left over supernatant DNA will be seen as white precipitate
sticking to the side. Dry for 10-15 min at 37 °C till there is no trace of solution IV.
10. Resuspend the pellet in 50μl of 1x TE, mix by tapping tubes with finger so that DNA goes
into the solution. Be sure mixing is complete with alternating tapping for 20 min.
11. Add RNAase A (optional)at the concentration of 50μg/ml incubate for 30 min at 37 °C
12. Add 3 µl of gel loading buffer to prepared DNA and 10-12µl of control DNA.
13. Load prepared DNA and control DNA samples in separate wells and electrophoresis in 0.8%
or 1% agarose gel to check the purity of your isolated DNA.

Lab session Three

Title:- Isolation of Genomic DNA

Objective:- To isolate Ggenomic DNA from Bacteria (E.coli).

Introduction

Generally, all methods involve the disruption and lysis of cells. This is followed sometimes by
the removal of RNA (by RNases, salt or other methods). Choosing which method will depend on
many selection factors including: DNA is isolated from proteins by several methods including
digestion of proteins by the enzyme proteinase K. Proteins are removed subsequently by salting-
out, organic extraction, or binding of the DNA to a solid-phase support (such as an anion-
exchange column or silica technology). DNA is finally recovered by ethanol/ isopropanol
precipitation.

In general, the separation of DNA from cells and cellular components have a common steps:

 Cell disruption/Lysis of Cell

 Removal of contaminants
 Recovery of DNA

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Materials

 TE buffer  phenol\chloroform (50:50)

 10% (w/v) sodium dodecyl sulfate  isopropanol

(SDS)
 70% ethanol
 20 mg/ml proteinase K
 3M sodium acetate pH 5.2

Procedure

1. 1. Grow E. coli culture overnight in rich LB medium.

2. Transfer 2 ml of the culture into a 2-ml micro centrifuge tube and spin 2 min then
decant the supernatant.
3. Resuspend the pellet in 467μl TE buffer by repeated pipetting.
4. Add 30μl of 10% SDS and 3μl of 20mg/ml proteinase K, mix, and incubate 1 hr at 37
°C.
5. Add 480μl of phenol/chloroform and mix well the DNA by inverting the tube until the
phases are completely mixed. Make it gently to avoid shearing
6. Carefully transfer the DNA/phenol mixture into fresh Eppendorf tube and spin at
12,000 RPM for 10 min.
7. Transfer the upper aqueous phase to a new tube and add 480μl of phenol/chloroform.
8. Again mix well and transfer to a fresh Eppendorf tube and spin 10 min.
9. Transfer the upper aqueous phase to a new tube.
10. Add 50μl of sodium acetate. Mix.
11. Add 700µl of ethanol and mix gently until the DNA precipitates.
12. Wash DNA by adding 1 ml of 70% ethanol and centrifuge 12000rpm for 60 sec.

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 13. Resuspend DNA in 200 μl TE buffer.
14. Store DNA at 4 ° C short term, -20 or -80 ° C long term.
15. After DNA has dissolved, determine the concentration and quality by measuring the
absorbance at 260 nm.

Lab session three (Kit method)

Gram Positive Bacterial Preparation

1. Prepare Lysozyme Solution using Lysozyme from chicken egg white, which is provided
in the kit. Prepare a 45 mg/ml stock solution of lysozyme as described under General Preparation
Instructions. 200μl of Lysozyme Solution is required per isolation procedure. Prepare extra
solution to account for pipetting error.

NOTE: (Optional) For higher yields - If working with Staphylococcus species, supplement the
Lysozyme Solution with 200 units/ml of lysostaphin. For Streptococcus species, supplement the
Lysozyme Solution with 250 units/ml of mutanolysin.

2. Harvest Cells
Pellet 1.5 ml of bacterial broth culture by centrifuging for 2 minutes at 12,000-16,000 x g
(≈13,000 16,000 rpm). Remove the culture medium completely and discard.

NOTE: If bacteria are grown in rich media such as terrific broth, it is necessary to reduce the
volume of the starting material of the overnight broth culture to 0.5 ml in order to avoid
overloading of the HiElute Miniprep Spin Column.
3. Resuspend cells
Resuspend the pellet thoroughly in 200μl of lysozyme solution (prepared in step 1b) and
incubate for 30 minutes at 37°C.
4. Lyse cells
Add 20μl of the Proteinase K solution (20 mg/ml) (MB086) to the sample. If residual RNA is not
a concern continue with step 5.
Optional RNase A treatment
If RNA-free genomic DNA is required, add 20 μl of RNase A Solution (DS0003), mix and
incubate for 5 minutes at room temperature (15-25°C), then continue with step 5.

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5. Add 200 μl of Lysis Solution (C1) (DS0010). Vortex thoroughly for few seconds and
incubates at 55°C for 10 minutes, then continues with step 6 (Prepare for binding).

NOTE: A homogeneous mixture is essential for efficient lysis.

6. Prepare for binding
Add 200 μl of ethanol (95-100%) to the lysate and mix thoroughly by vortexing for few seconds.

NOTE: A homogenous mixture is essential. A white precipitate may form on addition of

ethanol. It is essential to apply all of the precipitate to the HiElute Miniprep Spin Column. This
precipitate does not interfere with the DNA isolation procedure or with any subsequent
application. Do not use alcohols other than ethanol because this may result in reduced yields.

7. Load lysate onto HiElute Miniprep Spin Column (DBCA02)

Transfer the lysate obtained from step C onto HiElute Miniprep Spin Column provided.
Centrifuge at >6,500 x g (≈10,000 rpm) for 1 minute. Discard the flow-through liquid and place
the spin column in same 2.0 ml collection tube.

NOTE: Use a wide bore pipette tip to reduce shearing of the DNA while transferring contents
onto the column. It is essential to apply all of the precipitate to the column. If the solution has not
completely passed through the membrane, spin at a higher speed until all the solution has passed
through. Centrifugation at full speed will not affect the yield or purity of the DNA.

8. Prewash
Add 500 μl of Prewash Solution to the column and centrifuge at >6,500 x g (≈10,000 rpm) for 1
minute. Discard the flow-through liquid and re-use the same collection tube with the column.

9. Wash
(Prepare Wash Solution as indicated in General Preparation Instructions)
Add 500 μl of diluted Wash Solution (WS) (DS0012) to the column and centrifuge for 3 minutes
at maximum speed 12,000-16,000 x g (≈13,000-16,000 rpm). Transfer the column to new
collection tube and centrifuge again at same speed for the additional 1 minute to dry the column.
The column must be free of ethanol before eluting the DNA.

10. DNA Elution

Transfer the HiElute Miniprep Spin Column to new collection tube. Pipette 200 μl of the
Elution Buffer (ET) (DS0040) directly into the column without spilling to the sides.

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Incubate for 1 minute at room temperature. Centrifuge at >6,500 x g (≈10,000 rpm) for 1 minute
to elute the DNA.

NOTE: To increase the elution efficiency, incubate for 5 minutes at room temperature after
adding the Elution Buffer (ET), then centrifuge. Elution with volumes less than 200 μl increases
the final DNA concentration in the eluate significantly, but slightly reduces the overall DNA
yield. Storing DNA in water can cause acid hydrolysis.
11. Storage of the eluate with purified DNA:
The eluate contains pure genomic DNA. For short term storage (24-48 hrs) of the DNA, 2-8°C is
recommended. For long-term storage, -20°C or lower temperature (-80°C) is recommended.
Avoid repeated freezing and thawing of the sample which may cause denaturing of DNA. The
Elution Buffer will help to stabilize the DNA at these temperatures.

Lab session Four

Title:- Isolation of Genomic DNA From Plant

Objective:- To isolate Genomic DNA from the leaf of Nim plant

Introduction

Plant materials are among the most difficult cell structure for high quality DNA extractions. The
key is to properly prepare the tissues for extraction. In most cases this involves the use of liquid
nitrogen flash freezing followed by grinding the frozen tissue with a mortar and pestle. Liquid
nitrogen is difficult to handle and it is dangerous in an open laboratory environment such as a
classroom. For this reason the modified and very simple plant DNA extraction protocol is
designed to use fresh tissue.

Reagents and Buffers

CTAB Buffer A (EBA) Per 100 mL

 2% (w/v) hexadecyltrimethyl  100 mM Tris (pH 8.0) (Use 1 M stock) =

ammonium bromide (CTAB) = 2.0 g 10 mL

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 20 mM EDTA (Use 0.5 M stock) = 1  0.1% (w/v) ascorbic acid =0.1 g
mL  10 mM β-mercapto ethanol (BME)*
 1.4 M NaCl = 8.2 g (Use 14.3 M stock) =70 µL
 4% (w/v) polyvinylpyrrolidone (PVP) =
4.0 g

Extraction Buffer B (EBB) Per 100 mL

 50 mM EDTA(Use 0.5M stock)=2.5 ml  10 mM β-mercapto ethanol (BME)*

 100 mM NaCl = 0.6 g (Use 14.3 M stock) =70 µL

TE Buffer per 100 mL

 10 mM Tris (pH 8.0) (Use 1 M  1 mM EDTA (Use 0.5 M stock) =50

stock) =1.0 mL µL

Other Required Reagents

 20% (w/v) sodium dodecyl Sulphate  3 M sodium acetate (pH 5.2),

(SDS),  70% ethanol (stored -20oC) and
 5 M potassium acetate (Stored –  Absolute isopropanol (stored at -
20oC), 20oC)

Extraction Protocol

1. Grind the leaf using pastel and mortar.

2. Weight out 0.3 g of plant tissue
3. Immediately transfer tissue to a 1.5 ml micro centrifuge tube.
4. Once the sample is prepared add 300µl EBA, 900µl EBB and 100µl SDS.
5. Vortex and incubate at 65oC for 30 min.
6. Place tube on ice and add 410µl ice cold K Ac-. Mix by inversion back to ice for 30 min.
7. Centrifuge at 13,200 rpm for 15 min.
8. Transfer 1 ml of aqueous to a fresh tube, add 540µl of child absolute ethanol, and place in
ice for 20 min.

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 9. Centrifuge at 10,200 rpm for 10 min, discards the supernatant, dissolve the dry pellet in
250 µL of TE (if not put in water bath at 65 o C to facilitate dissolution) and add 250 µL
CAB buffer then incubate for 15 min at 65oC.
10. Add 1 volume of chloroform, centrifuge for 15 min and collect supernatant. (Repeat this
step for 1 time).
11. Add 540µl of child absolute Eth, place in 4 o C for 20 min, Centrifuge at 13,200 rpm for
15 min and Discard supernatant let pellet dry.
12. Resuspend the pellet and stored in 100µL of TE buffer, check the quality and purity your
isolated DNA.

Lab session Five

Title: DNA extraction Protocol from Animal cell

Objective: To isolate genomic DNA from whole blood

Introduction:

White blood cells (WBCs) are separated from a specimen of whole human blood (or from a
"buffy coat" specimen that has been separated from a whole blood sample) by mixing the
specimen with a hypotonic EDTA solution. The hypotonic solution lyses red blood cells, but
leaves WBCs intact. WBCs are separated by centrifugation, forming a WBC "pellet" in the
bottom of the centrifuge tube. The supernatant, containing hemoglobin, plasma proteins, and
other soluble components from the lysed red cells or plasma is poured off leaving a relatively
clean WBC pellet. The pellet is resuspended in the same hypotonic EDTA solution, and the tube
is again centrifuged and decanted, in order to further wash away contaminating proteins and
hemoglobin. The WBC pellet is resuspended in a small amount of NaCl solution, which helps
break up the pellet and disperse the cells. An SDS solution is added to the WBC/NaCl
suspension, which lyses the WBCs, releasing the DNA from the cell. The SDS also dissociates
protein/DNA complexes. RNase is added to the solution to destroy RNA. Remaining proteins
(including the RNase added in the previous step) are precipitated by phenol:chlorofrom:isoamyl
alcohol or high salt NaCl. The DNA is precipitated from the clean aqueous phase with isopropyl

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alcohol, washed with ethanol, and then dissolved in TrisEDTA solution. At this point the sample
is ready for quantitation, storage, and/or experimental use.

SPECIMEN:
The blood should be collected in vacutainers containing EDTA to prevent DNA degradation, and
should be refrigerated until processed. Up to 20 ml of blood may be processed in one tube in this
protocol. The approximate yields are 16-50μg DNA/ml whole blood. Optimum yields of DNA
are achieved when the blood samples are processed within five days of being obtained from the
patient. DNA may be obtained from samples collected in heparin or without anti-coagulants
(requires homogenization of clot), and/or samples that have been stored for longer than five days,
but the quality and yield may be reduced.

Preparation of Reagents
 TKM 1 Buffer / Low salt buffer (500 ml):

 0.605 g of TrisHCl (10mM)  0.372g of EDTA (2mM) was

pH 7.6, dissolved in 500ml of
 0.372 g of KCl (10 mM), distilled water
 1.016 g of MgCl 2(10 mM),

 Triton-X (10ml): Added 0.1 ml of 100 % Triton-X to 9.9ml of distilled water.

 TKM 2 Buffer / High salt buffer(100 ml):

 0.121 g of TrisHCl (10mM) pH 7.6,  0.074 g EDTA (2mM),

 0.074 g of KCl (10 mM),  0.467 g of NaCl (0.4 M) will be
 1.203 g of MgCl 2(10 mM), dissolved in 100ml of distilled water

 10% SDS: 10gram of sodium dodecyl sulphate will be dissolved in 100ml distilled water.
 6M NaCl: 8.765 g of NaCl was dissolved in 25 ml of distilled water.
 TE Buffer:

 0.030 g of TrisHCl (10mM) pH  0.009 g of EDTA (1mM) will be

8.0 dissolved in 100ml of distilled water

Procedure
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RBC Lysis
1. Take 300 µl of EDTA blood in an autoclaved 1.5 ml eppendorf tube
2. Add 900 µl of TKM 1 and 50 µl of 1x Triton-X.
3. Incubated at 370 C for 5 minutes to lyses the RBCs.
4. Centrifuge at 8000 rpm for 3 minute and discard the supernatant.
5. Repeat this step 2-3 times with decreasing amount of 1x Triton-X till, Until complete lyses
of RBC and a white pellet of WBCs remaining.
Cell Lysis
6. Add 300 µl of TKM 2 and 40 µl of 10% SDS to the cell pellet, Mix it thoroughly and
incubate at 370 C for 5 minutes.
7. At the end of incubation, add 100 µl of 6M NaCl and vortex it to precipitate the proteins.
8. Centrifuge at 8000 rpm for 5 minutes.

Precipitation of DNA
9. Transfer the supernatant in to a new eppendorf tube
10. Add 300 µl of isopropanol and inverting the eppendorf slowly to facilitate precipitation.
11. Centrifuge the eppendorfs at 8000 rpm for 10 minutes to pellet down the DNA.
12. Discard the supernatant; add 70% ethanol and mix slowly to remove any excess salts.
13. Centrifuge at 8000 rpm for 5 minutes to pellet down the DNA.
14. Discard the supernatant and air dry the DNA.
15. After thorough drying, 50 µl of TE buffer will be added to dissolve the
DNA and store it -20 oc for the next work.
16. Check the quality and purity of your isolated DNA.

Lab session Six

Title: - DNA Quality and Quantity Determination
Objectives:- To quantify and determine the purity of the extracted DNA samples using different
methods.
[

Introduction
Reliable measurement of DNA concentration is important for many applications in molecular
biology. The concentration of DNA in a sample and its condition are often estimated by running

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the sample on an agarose gel. Such concentration estimates are semi-quantitative at best and are
time consuming and confounded when numerous bands or a 'smear' of DNA are observed. For a
more accurate determination of the concentration of DNA in a sample, a UV spectrophotometer
at A260 wave length is commonly used. The purity of a DNA can be determined using a
comparison of the optical density values of the solution at various wavelengths.
Table 1. spectrophotometric Conversions
1 A260 unit (1-ml detection path) Concentration (µg/ml)
dsDNA 50
ssDNA 33
RNA 40
Oligonucleotide 20-30
The purity of DNA in a solution can be determined using a comparison of the optical density
values of the solution at various wavelengths. For pure DNA, the observed 260/280 nm ratio
will be near 1.8. Elevated ratios (> 1.8) usually indicate the presence of RNA, which can be
tested by running the sample, ~1µg, on an agarose gel. 260/280 ratios below 1.8 often signal the
presence of a contaminating protein or phenol. Alternatively, protein or phenol contamination is
indicated by 260/230 ratios greater than 1.5. Once you are sure your sample contains pure DNA,
an accurate determination of the concentration of DNA can be made.
6.1. DNA quantification by UV spectrophotometer
Materials:
 Sample DNA  Cuvettes
 Standard model spectrophotometer/  Pipettes, Pipettes tips and tip boxes
Nano drop.  Tissue paper
 Blank solution  Solid waste pot and Liquid waste pot
 Electrophoresis
Procedure
1. You will be provided all previously isolate of your samples from refrigerator
2. Start on spectrophotometer and keep it for 20 minutes
3. Auto-zero spectrum with blank of TE buffer for DNA/RNA or PBS buffer Protein
sample.
4. Withdraw buffer and rinse cuvette once with distilled water.
5. A pure DNA sample dissolved in TE buffer (pH 8.0) for stability, for which you will
determine an optical density (absorbance) spectrum using UV spectrophotometer.

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6. Adjust the wavelength that you will determine the absorbance readings at three wavelengths
(230, 260, and 280 nm). Measures the absorbance of all samples, and record the results in
Table 2. Using the wavelength ratios of these samples you will attempt to determine the
possible contaminants of the samples.
7. Graph the results to obtain an absorbance spectrum for (pure DNA).
8. Determine the absorbance readings for Sample 1, Samples 2, 3, and 4 at 230, 260, and 280
nm. Record the results in Table 2.
9. Determine the 260/280 and 260/230 ratios for each of samples and fill table 2 below.

Table 2. 260/280 and 260/230 Ratio Values for 4 Samples of DNA

Sample OD at 260 OD 280 OD230 260/280 260/230
1
2
3
4
10. Based on these ratios, how pure are the DNA samples you were isolated? If a DNA sample
is not pure, what are the possible contaminants of your sample? At which point in the
extraction procedure would these contaminants appear? What steps of the extraction
procedure would you need to change to get rid of these contaminants?
11. Determine the concentration of the DNA in solution assuming that you are dealing with pure
dsDNA.
 Hint: - For pure DNA, the observed 260/280 nm ratio will be near 1.8. Elevated ratios
usually indicate the presence of RNA, which can be tested by running the sample, ~1µg, on
an agarose gel. 260/280 ratios below 1.8 often signal the presence of a contaminating
protein or phenol. Carbohydrate contamination can be indicated by 260 /230ratios less than
1.5.

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6.2. DNA quantification using NanoDrop
Procedure
1. Switch on the instrument
2. After getting home page of programmes set default user and click on nucleic acid
3. Module Startup: When the software starts, you should see a message on screen having
two boxes “Ok” And “Cancel”: For best results, ensure measurement pedestal surfaces
are clean, load a water (1ul) sample onto the lower measurement pedestal and then
click ‘OK’. The message “Initializing Spectrometer- please wait” will appear. When this
message disappears, the instrument will be ready for use. All data taken will
automatically be logged in the appropriate archive file.
4. Set blank: Before making a sample measurement, a blank must be measured and stored
after making an initial blank measurement; a straight line will appear on the screen. For
the most consistent results, it is best to begin any measurement session with a blanking cycle.
This will assure the user that the instrument is working properly and that the
pedestal is clean. Follow the steps below to perform a blanking cycle:
5. Load a blank sample (1ul of the buffer, solvent, or carrier liquid used with your samples)
onto the lower measurement pedestal and lower the sampling arm into the ‘down’ position.
6. Click on the ‘Blank’ button.
7. When the measurement is complete, wipe the blanking buffer from both pedestals using
a laboratory wipe.
8. Quantify the Unknown DNA:
 With the sampling arm open, pipette the sample (1 ul DNA) onto the lower
measurement pedestal
 Close the sampling arm and initiate a spectral measurement using the operating
software on the PC. The sample column is automatically drawn between the upper
and
lower measurement pedestals and the spectral measurement made.
9. When the measurement is complete, open the sampling arm and wipe the sample from
both the upper and lower pedestals using a soft laboratory wipe. Simple wiping prevents
sample carryover in successive measurements for samples varying by more than 1000
fold in concentration.

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10. Measure subsequent samples. Repeat steps 5 and 6 for each sample.

6.3. Agarose Gel Electrophoresis for DNA Quality Analysis

Procedure
1. Prepare a 0.8% agarose gel.
2. Add 3l of 6X gel loading dye to 10 -12 l of each DNA sample before loading the wells of
the gel. Addition of dye allows us to note the extent to which the samples might have
migrated during electrophoresis, so that it can be halted at an appropriate stag
3. Load at least 1 or 2 wells with uncut, good quality DNA or any previously quantified DNA
samples (50ng and 100ng) as molecular weight standards.
4. Run the electrophoretic gel at 70V till the dye has migrated one-third of the distance in the
gel.
5. DNA can be visualized using a UV transilluminator and quantified in comparison with the
fluorescent yield of the standards.

Lab session Seven

Title-:PCR Amplification of DNA
Objective:-To familarize basic techniques of PCR
Introduction
The polymerase chain reaction (PCR) is a relatively simple technique that amplifies a segment of
DNA template to produce specific DNA fragments in vitro. Traditional methods of cloning a
DNA sequence into a vector and replicating it in a living cell often require days or weeks of
work, but amplification of DNA sequences by PCR requires only hours. Thus, PCR can achieve
more sensitive detection and higher levels of amplification of specific sequences in less time.
These features make the technique extremely useful, not only in basic research, but also in
commercial uses, including genetic identity testing, forensics, industrial quality control and in
vitro diagnostics.

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 Materials:
 sterile water
 10X amplification buffer with 15mM MgCl2
 10 mM dNTP
 50 μM oligonucleotide primer 1
 50 μM oligonucleotide primer 2
 5 unit/μl Taq Polymerase
 template DNA (1 μg genomic DNA, 0.1-1 ng plasmid DNA) in 10 μl

Procedure

1. Combine the following for each reaction (on ice) in a 0.2 or 0.5 ml tube: 
Master Mix preparation
No. 1X reaction (μl) 5X reaction (μl) 10X reaction (μl)
1. ddH2O 16.5 80.25 160.5
2. 10X PCR 2.5 12.5 25
buffer
3. 25mM MgCl2 2 10 20
4. DNTPS 1 5 10
5. Primer 1 0.625 3.125 6.25
6. Primer 2  0.625 3.25 6.25
7. Taq 0.2 1 2
Polymerase
8. Template DNA 1 10 20
2. Prepare a control reaction with no template DNA and an additional 10 μl of sterile water.
3. Run the following program.
Suggested Amplification Conditions
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 Stage Temperature (°C) Time (minutes) Cycles
Initial denaturation 94 7 1
Denaturation 94 1 34
Annealing 55 1 35
Extension 72 1 35
Final extension 72 3 1
Hold 4 Indefinite

Result???

Lab session Eight

Title:-Restriction Digestion

Objectives:- To cut /digest/ the isolated DNA samples at specific sites using restriction enzymes

Introduction

There are several things to consider. Digesting enough DNA; the DNA must be digested to
completion; i.e., no partial digests because they make interpretation difficult if not impossible
and the resolution of fragments.

 How much DNA to digest: - The big question, you may be digesting your DNA just to look
at it (an analytical gel) or to cut a band out of the gel for further treatment (a preparative gel).
Either way you want to be able to see the DNA bands under UV light in an Ethidium-stained
gel. Typically, a band is easily visible if it contains about 20 ng of DNA.

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  Too much DNA: - loaded onto a gel is a bad thing. The band appears to run fast
(implying that it is smaller than it really is) and in extreme cases can mess up the
electrical field for the other bands, making them appear the wrong size also.
 Too little DNA: - is only a problem in that you will not be able to see the smallest bands
because they are too faint. Having said all that, DNA gels are forgiving and a wide range
of DNA loads will give acceptable results.
 Buffer: - This is the 10x buffer that comes with the restriction enzyme. Most companies have
about four different kinds of buffer (A, B, C, D or 1, 2, 3, 4) and occasionally a "unique
buffer" for a particular enzyme. Do not worry about this. Many restriction enzymes are
forgiving about buffers. EcoRI, for example, works excellently in all four of the standard
buffers. Fundamentally it is a question of salt concentration (high or low) particularly,
Magnesium concentration and pH.
 H2O:- Standard distilled water. It is always good to add the buffer and water into the tube
first. If you put the enzyme in straight on top of the buffer then it may become irreversibly
denatured.
 Enzyme: - 0.5µl of enzyme is plenty for a miniprep digestion. Do not use more enzyme than
10% of the final reaction volume.
 Pre-mixes:- If you are doing many digests then it may advisable to make up a pre-mix in
order to save on pipetting. Suppose you are doing 16 digestions all with the same enzyme, on
16 different DNA samples. You should set up a 17x pre-mix (always do 1 extra to make up
for slight pipetting inaccuracies) containing buffer, water, and enzyme.
 Double digests: - You may be digesting your DNA with two (or more) enzymes. This is fine
but you have to make sure to use the buffer that will be most compatible with all the
enzymes.
 Special conditions: - A few enzymes require special conditions. It will say in the catalogue.
Some require BSA (bovine serum albumin) added into the mixture. Some require weak
detergents (triton-X-100) to reduce surface tension. Some require to be incubated at
temperatures other than 37°C (Ex. 50°C).
 Star activity: - This is when the enzyme cuts at sites other than its cognate element. Eg.
EcoRI is supposed to only cut GAATTC. But under extreme conditions, it might possibly
cut CAATTC also.

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Chemicals and Regents

 Restriction enzymes of choices, such  100 % Ethanol

as BamH1 and HIndIII  3 M Sodium acetate (pH 5.2)
 Restriction enzyme reaction buffer  Genomic DNA isolated from
 Double Distilled water bacteria or other source
 70 % Ethanol

Procedure

No. Components Amount to be added For one reaction

1. Distilled water 20 µl
2. 10 X restriction enzyme reaction buffer 3 µl

1 reaction
3. DNA (at a concentration of 0.5-2 µg/ µl) 5 µl
4. BamHI (10 µg/ µl) 1 µl
5. HIndIII (10 µg/ µl) 1 µl
Total volume 30 µl
1. Set up the following reactions (3x) volumes for (BamHI, HIndIII and sample control).
2. Mix gently and spin for 2 seconds in a micro centrifuge.
3. Incubate at an appropriate temperature (37 °C) for 30 minutes.
4. Add 3 µl of the sodium acetate and 90 µl of 100 % ethanol.
5. Store the tubes at -20 °C for 1 hour.
6. Spin in a micro centrifuge at maximum speed for 15 minutes.
7. Discard supernatant and add 400 µl of 70 % ethanol.
8. Spin in a micro centrifuge at maximum speed for 5 minutes.
9. Discard the ethanol and add another 400 µl of 70 % ethanol.
10. Spin in a micro centrifuge at maximum speed for 5 minutes.
11. Withdraw and discard as much liquid (ethanol) as possible then air-dry the pellet.
12. Resuspend the pellet in 20 µl of distilled water.
13. Keep at 4 °C or -20 degrees °C and examined later by agarose gel electrophoresis.
 Tips: DNA should be stored at -20°C, but repeated freeze-thaw should be avoided. So
if it is needed frequently or in a couple of days stores it at 4 °C.

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 Result:

After agarose gel electrophoresis, fragments of different base pair size will be visible on the
agarose gel. For the controls without restriction enzyme, the nucleic acid yields larger bands base
pairs.

Lab session Nine

Title: - Agarose Gel Electrophoresis

Objectives: - To separate DNA fragments based on their size

Introduction

The aim of this gel electrophoresis is to separate fragments of nucleic acids (DNA and RNA) and
to detect them by staining with ethidium bromide and visualizing under UV light. The separation
process is facilitated by electric current and based on molecular weight and charge. The most
commonly used media in horizontal gels is agarose at different gel strength.

Strength of the Agarose gel for Different Size DNA Fragments

DNA Fragment Size (KBp) 5-50 1-25 0.7- 10 0.5 – 10 0.2- 5 0.1-2 0.02-1

Agarose Concentration (%) 0.3 0.5 0.8 1 1.5 2 2.5

Materials and Chemicals

 Microwave  Micropipette and Pipette tips

 Balance (10, 100, 1000 µl)
 Electrophoresis’ chamber and  Agarose powder
power supply  Buffer such as TBE or TAE
 Transluminator/ gel (approximately 0.7-0- 1%)
documentation system  Gel loading dye
 Erlenmeyer flasks  10µg/ml ethidium bromide
 Gloves

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Procedure:
1. Seal the edges of a clean, dry glass plate with a tape to form a mould.

2. Weigh the appropriate amount of agarose powder and add in a clean 500 ml glass bottle.

3. Heat up the slurry in microwave (until bubbles appear).

4. Wear an oven mitten and carefully swirl the bottle or flask.

5. Use insulated gloves to transfer the flask/ bottle into water bath at 55 oC.

6. While the agarose gel is cooling, add appropriate amount of EtBr and mix well, choose the

appropriate comb to form appropriate walls.

7. Pour the cooled agarose into the gel tray (mould) and wait till it solidifies.

Hint: The gel should be between 3 mm- 5 mm thick. Check that no air bubbles are under or
between the teeth of the comb. Air bubbles present in the molten gel can be removed easily
by poking them with a tip.
8. Remove the tape and the two separating rulers and mount the tray in the electrophoresis tank.

9. Mix the samples with loading dye up to a volume of 12-15µl.

10. Slowly load the sample mixture into the slots of the submerged gel using a micropipette.

11. Close the lid of the gel tank and attach the electrical flexes DNA will migrate.

12. At the end of the run, turn off the current and remove the flexes and the lid from the tank.

Hint: The migration of the loading dye depends from the type of agarose and its concentration as
well as from the running buffer used (TBE or TAE).
Hint: The presence of ethidium bromide in the gel allows it to be examined by UV illumination
at any stage during the electrophoresis. The gel tray may be removed and placed directly on the
transluminator. Alternatively, the gel may be examined using a hand held source of UV light. In
either case, turn off the power supply before examining the gel. During the electrophoresis, the
ethidium bromide migrates towards the cathode (in opposite direction to that of the DNA). This
can lead to loss of significant amounts of ethidium bromide from the gel, making detection of
small fragments difficult. In this case staining the gel by immersion in buffer containing
ethidium bromide is recommended. Also, having a separate ethidium bromide tank reduces the
risk of contamination.
13. Examine the gel and photograph under UV light.

Lab session Ten

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Title: Transformation of E. coli Cell
Objectives

 Understand recombinant techniques and the transformation procedure using the heat
shock method.
 Understand how we can screen for a gene of interest and the importance of marker or
reporter genes in molecular biology experiments.
 Investigate how DNA can be transferred to another organism and the change in
phenotype (physical characteristics) that may result from such a transfer.
 Learn the importance of the sterile techniques that are used to handle bacteria and the
decontamination necessary when the experiment is complete.

Introduction

In molecular biology, transformation refers to a form of genetic exchange in which the genetic
material carried by an individual cell is altered through incorporation of foreign (exogenous)
DNA. This foreign DNA may be derived from unrelated species and even other kingdoms, such
as bacteria, fungi, plants or animals.

E. coli is not naturally transformable, the ability to take up DNA or competency must be induced
by chemical methods using divalent and multivalent cations (calcium, magnesium, and
manganese, rubidium, or hexamine cobalt).  Alteration in the permeability of the membranes
allows DNA to cross the cell envelope of E. coli which is composed of an outer membrane, an
inner membrane, and a cell wall.  The outer membrane of E. coli can be understood by
application of the fluid mosaic model for membranes and is composed of phospholipids,
proteins, and lipopolysaccharides.  Many channels or zones of adhesions are formed by the
fusion of the outer membrane and the inner membrane through the cell wall layer.  Although the
transformation mechanism is not known, previous studies indicate that these channels allow for
the transport of DNA molecules across the cell membrane.    
The negative charges of the incoming DNA, however, are repelled by the negatively charged
portions of the macromolecules on the bacterium’s outer surface. The addition of CaCl 2 serves to
neutralize the unfavorable interactions between the DNA and the polyanions of the outer layer. 
The DNA and competent cells are further incubated on ice for thirty minutes to stabilize the lipid

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membrane and allow for increased interactions between calcium ions and the negative
components of the cell. The reaction mixture is then exposed to a brief period of heat-shock at
42oC.  The change in temperature alters the fluidity of semi-crystalline membrane state achieved
at 0oC thus allowing the DNA molecule to enter the cell.

Procedure for Competent Cell Formation

1. Grow a 5mL overnight culture of LB broth at 37°C with vigorous shaking.

2. Take 2ml of this culture to a new Eppendorf tube.
3. Cool cells on ice for 10 min and spin in centrifuge for 5 minutes, 4°C, at 3,000 rpm.
4. Discard the supernatant and quickly put cells back on ice
5. Resuspend the cells in 2mLs of ice cold (sterile) CaCl2 (50mM) and leave on ice for 15
minutes
6. Centrifuge cells and discard the supernatant then resuspend cells in 500 μl CaCl2. Use
immediately for transformation or store at -80 °C. If cells used for transformation are frozen,
remove tube from freezer and immediately put on ice. Allow these cells to thaw on ice,
which takes about 30 minutes.

Transformation Procedure for blue/white screening

1) Remove cells from the -80 °C freezer and thaw on ice.

2) Add 100 μl of cells to an eppendorf containing the appropriate DNA (5 μl )

3) Incubate on ice for 30 minutes.
4) Heat shocks the cells at 37 °C for 2 minutes.
5) Place the eppendorfs on ice for 1 minute.
6) Add 1 ml of room temperature LB media to each eppendorf.
7) Shake the tubes for 1 hour at 37 °C .

8) Prepare the X-Gal plates by spreading the following: 60 μl LB media, 40 μl X-Gal (20

mg/ml) and 4 μl 100mM IPTG .μ

9) Plate the bacteria onto the X-Gal plates and place at 37 °C overnight and observe the
result in the next day.
10) Extra cells should be frozen in dry ice and then moved to the -80 °C freezer

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Transformation of the bacterium E. coli using a gene for Green Fluorescent Protein

Introduction

In this lab, Green Fluorescent Protein (GFP) from the bioluminescent jellyfish Aequorea victoria
will be incorporated into a plasmid along with a gene for resistance to the antibiotic ampicillin.
The GFP is actually located in discrete spots around the bell margin of the jellyfish and will
fluoresce under certain conditions. When inserted into a plasmid and used for the transformation
procedure, the transformed bacteria will express their newly acquired jellyfish gene and produce
the fluorescent protein, which causes them to glow green under ultraviolet light. The mutant
form of GFP used in pGREEN makes the bacteria a yellow-green color even in white light.
This plasmid contains an ampicillin-resistance gene in addition to the GFP gene. Ampicillin is an
antibiotic and works by preventing E.coli from constructing cell walls, thereby killing the
bacteria. When the ampicillin resistance gene is present it directs the production of an enzyme
that blocks the action of the chemical and the bacteria are able to survive. Bacteria without the
plasmid and hence the resistance gene are unable to grow on a plate containing ampicillin in the
medium and only the transformants will survive.

Materials

For each lab group

 Gloves  disposable inoculating loops

 Safety goggles if desired  Foam cup with crushed ice
 Waterproof marker  2 LB plates
 Microtube rack  2 LB + Amp plates
 disposable pipettes

Groups Will Share:

 70% Ethanol  Luria broth

 50 Mm CaCl2(keep on ice)

Common materials

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  Micropipette  Distilled water
 Water bath (with floating tube rack)  37°C incubator and 42°C
 Streak plates of E.coli  Parafilm
 pGLO plasmid  Waste container
 Crushed ice

Procedure

Day 1:

1. Make sure that you have all of the materials before starting the lab
2. Label one closed micro test tube +pGLO and another –pGLO.
3. Add 250 µl of 50mMCaCl2 ice cold transformation solution to each tube (“+” and “-”)
and immediately place both on ice.
4. Use a sterile loop to pick up a single colony of bacteria from your starter plate. Be careful
not to scrape off any agar from the plate. Pick up the +pGLO labelled tube and immerse
the loop into the CaCl2 solution (transforming solution) at the bottom of the tube. Spin
the loop between your index finger and thumb until the entire colony is dispersed in the
solution. Place the tub back in the ice.
5. Transfer 10 L of the 0.08 g/L pGLO plasmid solution directly into the E. coli cell
suspension in the +pGLO tube. Tap tube with a finger to mix. Do not add the pGLO
plasmid solution into the –pGLO tube.
6. Incubate the tubes on ice for 10 minutes.
7. While the tubes are sitting on ice, label each of your four agar plates on the bottom (not
the lid) with your group number and the date; then as follows:  Label one LB/amp plate
+pGLO  Label the LB/amp/ara plate +pGLO  Label the other LB/amp plate –pGLO 
Label the LB plate –pGLO

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8. Following the 10 minute incubation at 0°C, heat shock the cells in the +pGLO and –
pGLO tubes into the water bath, set at 42°C for 90 seconds. Three fourths of the tube
should be under water.
Optional: For best transformation results, the change from 0°C to 42°C then back to 0°C
must be rapid.
9. After heat shock, immediately return the tubes to ice at least two minutes.
10. Remove the tubes from the ice and transfer 250 L of LB nutrient broth to the +pGLO
tube. Close the tube and gently tap with your finger to mix. Repeat with a new
micropipette tip for the –pGLO tube. Incubate each tube for 10 minutes at room
temperature. Make sure that you add to the "-" tube first so as to avoid cross-
contamination of the plasmid.
11. Transfer 100 L of the transformation and control suspensions onto each of the four
labelled plate.

12. Spread the suspensions evenly around the surface of the agar. Allow plates to set for 10
minutes.

1. Covering with Parafilm to seal the lids. Stack your plates and tape them together. Place
the plates upside down in an incubator at 37oC or in 48-72 hours at room temperature.
The results will be ready to observe in 24 hours if incubated

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 2. After tips and tubes have sit in bleach solution for at least 15 minutes, pour liquid in
bacterial waste container down drain. Collect tips and tubes in a plastic bag and discard in
the normal garbage. Spray down workspace with bleach/ 70% ethanol solution. Wash
hands before leaving lab.

Day 2
 Retrieve your plates from incubator or other storage area and check for
growth.

Student Activity POST-LAB QUESTIONS

1. What color was the pGLO plasmid DNA when you exposed it to the UV light?
Will all of the plates have bacteria growing on them?
2. On which of the plates would you expect to find bacteria most like the untransformed E.
coli colonies you initially observed? Explain your prediction.
3. If there are any genetically transformed bacterial cells, on which plate(s) would they most
likely be located? Explain your prediction.
4. Shine the UV light on the plate with the transformants. What color are the bacteria?
Compare this answer to what you observed when the light was shined onto the pGLO
plasmid. Is there a difference? Why?
5. What is meant by control plate(s)? What was the function of each plate that you set up?

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 6. To genetically transform an entire organism, you must insert the new gene(s) into every
cell in the organism. Which organism is better suited for total genetic transformation—
one composed of many cells, or one composed of a single cell? Why?
7. Describe how you could use two LB nutrient agar plates, some E. coli, and some
ampicillin to determine how nontransformed E. coli cells are affected by ampicillin.
8. From the proposed experiment in question 7, what would you expect your experimental
results to indicate about the effect of ampicillin on the E. coli cells?

Lab session Eleven

Title: Separation of protein by SDS PAGE

Objectives:

 Separation of protein fractions using SDS-PAGE

 Determine their molecular weight of separated proteins

1. Introduction
SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) is probably the world’s most widely
used method. The method involves the separation of protein based on their size. By heating the
sample under denaturing and reducing condition, protein become unfolded and coated with SDS
detergent molecules, acquiring a high negative charge that is proportional to the length of the
polypeptide chain. When loaded onto a gel matrix and placed in an electric field, the negatively
charge protein molecules migrate towards the positively charge electrode and are separated by a
molecular sieving effect. After visualization by a protein specific staining technique, the size of
protein can be estimated by comparison of its migration distance, with that of a standard of
known molecular weight. It is possible to blot the separated protein onto a positively charge
membrane and to probe with protein specific antibodies in a procedure termed western blotting.

2. Materials Required
2.1. Acrylamide(30% stock):- dissolved 29.2 g acrylamide and 0.8 bis-acrylamide in distilled
water and make upto 100ml. Store under dark in amber colour bottle at 4°c(can use upto 3
month). Caution. Acrylamide is a neurotoxin. Always wear gloves, safety glasses, and
a surgical mask when working with acrylamide powder.

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2.2. Resolving gel/ separating gel buffer pH 8.8, 1.5M tris-HCl: - dissolved 18.17g tris in 75
ml distilled water. Adjust to pH 8.8 with 6 N HCl. Adjust the total volume to 100ml with
distilled water and store at 4°c.
2.3. Stacking gel buffer, pH-6.8, and 1.0Mtris-HCl:- dissolved 3g tris in 40 ml distilled
water, adjust to pH 6.8 with 6N HCl. Adjust the total volume to 50ml with distilled water.
Store at 4°C.
2.4. Electrophoresis buffer pH 8.3:- dissolved 3g or (25mM) tris, 14.4 g or (250mM)
glycine and 1g SDS in 100ml of distilled water. Store at 4°C.
2.5. Ammonium per sulphate-initiator 10%:- dissolved 0.1 g APS in 1 ml distilled water.
2.6. TEMED (NNN’N’ Tetramethylenediamine):-catalyst.
2.7. Sample buffer:- 7.25 ml distilled water + 1.25 ml stacking gel buffer + 1ml glycerol + 0.5
ml β- mercaptoethanol + 150 mg SDS and a pinch of bromophenol blue.
2.8. Staining solution: -dissolved 200mg coomassie brilliant blue R in 50ml methanol/ethanol,
7ml acetic acid and 43ml distilled water & filter it.
2.9. De-staining solution:- add 7ml acetic acid to 30ml methanol/ethanol and 63ml distilled
water.
2.10. Vertical slab-gel electrophoresis equipment.
2.11. Acrylamide mixture (10%) for 25 ml of resolving gel:-9.9 ml, distilled water + 8.3ml,
0% acrylamide + 6.3ml, 0.5 M tris-HCl + .25ml, 10% SDS + .25ml, 10% APS +.01 ml,
TEMED.
2.12. 5% stacking gel for 5 ml:- 3.04ml, distilled water + 0.83ml, 30% acrylamide + .63ml,
0.5M Tris – HCl(pH-6.8) + 0.05ml, 1% SDS +0.05ml, APS + 0.005ml, TEMED.
Tip: gel buffer and self-prepared acrylamide/bisacrylamide stock solution should be
filtered, degassed and stored at 4°C.

3. Sample Preparation
3.1. Take a protein sample in to Eppendorf tube.
3.2. Add 500μl of TE in the tube and dissolve the tube by gentle shaking.
3.3. Then add same volume of sample buffer.
3.4. Finally heat the sample on boiling bath for 5 minutes and then immediately keep on ice.

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 4. Preparing acrylamide gels: Procedures: Gel Casting Tray
4.1. Assemble glass plates and spacers in gel casting apparatus. Tip:-the plate should be
thoroughly cleaned and dried before use.
4.2. Seal the glass plates on 3 sides with 1% agarose, then mix the components for the
resolving gel
4.3. Pour the separating gel/ resolving gel mixture to a level of approximately 2 or 2.5 cm
below the top of the shorter plate,
4.4. Pace a layer of 250μl DDI H2O over the top of the resolving gel to prevent meniscus
formation in the resolving gel. Allow resolving gel to polymerize up to 30 min at room
temperature.

Tips: prepare the solution freshly each time it is required. As soon as ammonium persulphate is
added, the gel should be poured quickly before the acrylamide polymerizes.

4.5. After polymerization remove water from the top of resolving gel through draining.
Rinse with DDI H2O, drain, and wick any remaining DDI H2O.
4.6. Mix components for stacking gel.
4.7. Pour stacking gel solution over the resolving gel/ separating gel, so that gel plates are
filled.
4.8. Immediately insert a comb and allow polymerizing the gel.

Resolving Gel Preparation Stacking Gel Preparation

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 5. Sample Loading
5.1. Prepared the required volume of sample (100mg protein per lane) + equal volume of
sample buffer.
5.2. Heat the sample in boiling water bath for 5 min to denature the protein. Immediately keep
them on ice to retain the denature stage.
5.3. Remove the comb from the mould; wash the well with distilled water. Tip: with a marker
pen mark the number or the position of the wells before removing the comb. This aids
easy loading of sample.
5.4. Mount the gel on electrophoretic apertures’.
5.5. Add electrophoresis buffer to the top and bottom reservoir of the electrophoretic
apparatus.
5.6. Load the sample along with marker protein into the wells (20 μl)

6. Electrophoresis/ Running the gel

6.1. Attach the apparatus to the power supply unit and apply 8 V/Cm for stocking gel (70V)
and 15 V/Cm for resolving gel(150-200V)
6.2. Electrophoresis is continued until bromophenol blue reaches the bottom of the gel.
6.3. Dismantles apparatus and remove gel from between the plates and place in a tray
containing distilled water cut a small corner of the gel to indicate the direction of loading.

7. Coomassie Staining
7.1. Immerse the gel in 5 volume of staining solution (Methanol, CBB, Glacial acetic acid and
Water) and stain for 4 hrs.at room temperature with gentle shacking. Coomassie brilliant
blue R reacts non-specifically with proteins.
7.2. Gently agitate the stained gel in destining solution (methanol, Glacial acetic acid and
Water)) until the background becomes clear.
7.3. Store in 7 % acetic acid. Visualize the band in an illuminator.

END OF THE COURSE

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