Bacterial Cloning

Bacterial cloning inside a plasmid involves introducing a gene or DNA fragment into a plasmid
vector, which is then introduced into bacteria (usually E. coli) for replication and possibly
protein expression. Here is a step-by-step breakdown of the process:

1. Plasmid Isolation
2. Genomic DNA isolation
3. PCR of both insert (genomic DNA) and vector (plasmid)
4. Agarose gel electrophoresis
5. PCR purification
6. Restriction digestion of PCR purified product
7. Agarose gel electrophoresis of digested product
8. Gel purification
9. Ligation reaction
10. Transformation
11. Media transfer
12. Plasmid isolation
13. Colony PCR
14. Restriction digestion (to confirm cloning)

1. Plasmid isolation:
(SPINeasyTM Plasmid Miniprep Kit)
Quick-Start Protocol

Notes before starting:

 Briefly spin down the vial of RNase A and add the entire solution to Resuspension Buffer
SN1. Mark the bottle and store Resuspension Buffer SN1 with RNase A in 2-8 °C.
 Add 9 mL (0.9 mL for sample kit) isopropanol to Wash Buffer NW1 and mark the bottle.
 Add 50 mL (5 mL for sample kit) absolute ethanol to Wash Buffer NW2 and mark the
bottle.
 Prepare two labelled 1.5 mL microcentrifuge tubes per prep: one for sample lysis and
another for elution of purified plasmid.
 Centrifugation speed stated in the manual will be a guideline; use the maximum speed
available if 14,000 g is not feasible.

Culture:
 Inoculate bacteria into 1-5 mL LB medium containing the appropriate selective antibiotic.
 Incubate overnight (approximately 16 hours) at 37 °C with vigorous shaking at 180-250
rpm.
 Harvest the cells by centrifugation for 5 mins @ 5,000 g or for 3 mins @ 10,000 g. Discard
the supernatant.
 Column preparation
 Pipette 200 μL Equilibration Buffer into Column SN with collection tube. Incubate for 1
min at room temperature and centrifuge for 30 sec @ 14,000 g. Discard flow through and
reuse collection tube.
 Keep the columns aside for later use.

Lyse:
 6. Resuspend bacterial cell pellet in 250 μL Resuspension Buffer SN1. If the cells are in a
culture tube, transfer the suspension to a microcentrifuge tube.
 7. Add 250 μL Alkaline Lysis Buffer SN2 and mix well by gently inverting the tube several
times. Do not vortex or pipette vigorously.
 8. Incubate at room temperature for 2 mins. Do not exceed 5 mins.
  Bind
 9. Add 350 μL Neutralization Buffer SN3 and mix well by gently inverting several times.
Do not vortex or pipette vigorously.
 10. Centrifuge for 10 mins @ 10,000 g.
 11. Transfer all of the lysate supernatant to a Column SN with collection tube.
 12. Centrifuge for 1 min @ 14,000 g. Discard flow through and reuse collection tube.

Wash:
 13. Add 500 μL Wash Buffer NW1 to the column.
 Centrifuge for 1 min @ 14,000 g. Discard flow through and reuse collection tube.
 Add 750 μL Wash Buffer NW2 to the column. Incubate at room temperature for 1 min. 16.
Centrifuge for 1 min @ 14,000 g. Discard flow through and reuse collection tube.
 Centrifuge for an additional 1 min @ 14,000 g to dry column.
(Optional: Incubate at 55 °C for 3-5 mins to dry column completely.)

Elute:
 18. Remove collection tube and place column into a clean 1.5 mL microcentrifuge tube. 19.
Add 50 μL Elution Buffer SN to the center of the membrane. Incubate at room temperature
for 1 min.
 20. Centrifuge for 1-2 mins @ 8,000 g to elute plasmid DNA.
 21. Eluted plasmid DNA will be collected in the microcentrifuge tube.

2. Genomic DNA isolation:

Using Kit manual

3. Polymerase Chain Reaction (PCR):

Polymerase Chain Reaction (PCR) is a molecular biology technique used to amplify specific
segments of DNA. It allows for the rapid and selective replication of a target DNA sequence,
making millions of copies from a small initial sample. PCR follows three primary steps that are
repeated for several cycles, each step occurring at a different temperature:
Denaturation (94–98°C): The double-stranded DNA is heated to separate it into two single
strands by breaking the hydrogen bonds between base pairs. This step typically lasts around
20–30 seconds.
Annealing (50–65°C): The temperature is lowered to allow primers (short single-stranded
DNA sequences) to bind to their complementary sequences on the target DNA. The annealing
temperature depends on the primer sequences and usually takes 20–40 seconds.
Extension/Elongation (68–72°C): DNA polymerase (commonly Taq polymerase) synthesizes
new DNA strands by adding nucleotides to the primer in the 5' to 3' direction. The ideal
temperature for Taq polymerase is around 72°C.

Key components:
1. Template DNA (gDNA)- 2.5ul
2. Forward primer- 2.5ul
3. Reverse primer- 2.5ul
4. dNTP- 1ul
5. Buffer Taq- 5.6ul
6. Nuclease-free water- 34.9ul

Protocol:
 Lebel every PCR tube.
 Take template DNA (gDNA) in a PCR tube.
 Add forward primer and reverse primer.
 Add dNTP, Buffer Taq (NEB) and nuclease-free water.
(Note: all the components should be taken out in ice and the enzyme in ice box)
 After adding all the components place the PCR tubes in cool box
  Set the PCR machine (for 35 cycles)
- 5mins denaturation step at 94°C
- 94°C for 30sec
- 70°C for 30sec (Annealing step)
- 72°C for 30sec
- Final 1min extension step at 72°C
- Hold at 4°C
 Place the PCR tubes carefully.
 Close the lid.
 Start the machine.

4. Agarose gel electrophoresis:

The principle of agarose gel electrophoresis is based on the separation of DNA (or RNA)
fragments according to their size and charge. DNA molecules are negatively charged due to
their phosphate backbone, so when an electric field is applied, they move towards the positive
electrode (anode) through the agarose gel matrix. The agarose gel acts as a sieve, with smaller
DNA fragments migrating faster and farther through the gel, while larger fragments move
more slowly.

Preparation of 0.8% Agarose gel:

Key components:
1. Agarose powder
2. 1X TAE buffer
3. EtBr
4. Gel apparatus

Protocol:
 Weigh 0.8g of agarose powder in a conical flask and add 100 ml of TAE buffer
 Heat the mixture carefully until it become a clear mixture
 Cool down the mixture
 Add 3ul of EtBr when the conical flask is touchable carefully
 In the mean time set the casting tray
 After adding EtBr, immediately pour the gel mixture in the casting tray
Note: after adding EtBr the vapour should not be inhaled while pouring also, as EtBr is
carcinogenic
 Within 30-45min gel should be set
 Carefully remove the comb
 Take out the DNA ladder in ice and take 5ul of the ladder using micropipette and load in
a well
 Taka a paraffin, add 2ul of loading dye and mix 2ul of PCR product with it, take the
whole mixture with micropipette and load into the well
 Set the machine at 150mV for 30min
 After 30min take out the gel and see the bands under UV to confirm the PCR products

5. PCR purification:
Using Kit manual

6. Restriction digestion:
Restriction digestion is crucial in molecular cloning because it allows for precise cutting of
DNA at specific sequences using restriction enzymes, generating compatible sticky or blunt
ends. This enables the insertion of DNA fragments into plasmid vectors at defined locations.
The vector and plasmid both should be cut with same restriction enzymes.

Key components:
1. PCR purified products
 2. Two restriction enzymes
3. Cut smart buffer
4. Autoclaved water

Protocol:
 Take out the restriction enzymes and cut smart buffer in cool box
 Add PCR purified insert, restriction enzymes, cut smart buffer and autoclaved water in a
1.5ml eppendorf and same with the PCR purified vector
 Keep the eppendorf at 37°C for 2 hours

7. Agarose gel electrophoresis of digested products:

Follow step 4. Run the PCR purified products simultaneously with the digested products to
confirm the digestion.

8. Gel purification:
Cut the gel bands carefully using the UV light.
Purification-using Kit manual

9. Ligation reaction:
The principle of ligation involves the covalent joining of DNA fragments using the enzyme
DNA ligase. DNA ligase catalyses the formation of phosphodiester bonds between the 3'-
hydroxyl and 5'-phosphate ends of adjacent DNA strands. This process effectively stitches
together the sticky or blunt ends of DNA fragments, allowing for the creation of continuous,
recombinant DNA molecules. The ligation reaction relies on the presence of ATP (or similar
energy sources) in the buffer to drive the formation of these bonds, facilitating the assembly of
DNA constructs for various applications in molecular cloning and genetic engineering.

Key components:
1. Gel purified products
2. T4 DNA ligase

Protocol:
 Add gel purified insert and vector with the T4 DNA ligase in a 1.5 eppendorf.
 Keep the entire mixture in X°C for overnight

10. Transformation:
The recombinant plasmid is introduced into competent bacterial cells (e.g., E. coli) through a
process called transformation using heat shock method.

Key components:
1. Cloning vector (E. coli)
2. Ligated product

Protocol:
 Take out the E. coli strain from the freezer and mix it with the ligated product and keep
the eppendorf in ice for 30 minutes
 Set the water bath at 42°C
 After 30 minutes hold the eppendorf in the water bath for 30 sec
 After 30 sec immediate place the eppendorf in ice for 20min
 Take the agar plate
 Sanitize the desk, agar plate cover and spreading rod with alcohol and heat
 After 20min of ice incubation take out the whole mixture using a micropipette
 Place drop wise over the agar plate
 Spread the drops using spreading technique
 Keep the agar plate upside down at 37°C incubator overnight
11. Media transfer:
Protocol:
 LB broth preparation:
i. Weigh n gm of LB powder and add n ml of distilled water in conical flask
ii. Cover it up with cotton ball and foil
iii. Autoclave the conical flask
 Under the laminar flow, carefully open the cotton ball and foil, and add n ul of
Ampicillin
 Take out the observed the colony using micropipette tip and put it into the lb media under
the laminar flow
 Keep the conical flask at shaker incubator at 37°C for overnight

12. Plasmid isolation:

Using Kit manual

13. Colony PCR:

Follow step 3.

14. Restriction digestion:

Follow step 6. Do restriction digestion of both parent plasmid and cloned plasmid.

15. Agarose gel electrophoresis of digested product to confirm clone:

Follow step 4. Run the gel of both parent plasmid and cloned plasmid simultaneously.
 SDS-PAGE (Sodium Dodecyl Sulphate- Poly
Acrylamide Gel Electrophoresis)
The principle of SDS-PAGE is based on separating proteins primarily according to their
molecular weight. It utilizes the denaturing detergent SDS to ensure that proteins are uniformly
charged and eliminates the influence of their native structure or charge differences during
electrophoresis.

 Preparation of gel:
Key elements:
1. Acryl mix
2. 1M Tris (pH 6.8 for stacking gel, pH 8.7 for resolving gel)
3. 10% SDS
4. Autoclaved water
5. 10% APS
6. Temed
7. Isopropanol

Protocol:
 Wash the notched alumina & rectangular glass plates thoroughly and dry them.
 Insert the alumina plate, and glass plate one by one into the polythene pouch. (The notch of
the alumina plate should be facing the top.)
 Insert a spacer in between the notch and rectangular plates. Move to the edge.
 Insert another spacer and move to the other side.
 Clamp this arrangement onto the gel casting stand with the dummy plate.
 Prepare the following separating gel mix (12%) in a small vial:

Component For 1 gel For 2 gel

Acrylamide monomer 2.800ml 5.600ml
4x separating gel buffer (pH 8.7) 1.750ml 3.500ml
10% SDS 0.070ml 0.140ml
10% APS 0.035ml 0.070ml
D. water 2.345ml 4.690ml
Final volume for mini gel 7.0ml

 Degas the solution for 5 minutes and add TEMED 7ul. (14μl for 2gel)
 rotate the flask gently and pour the gel mix into the polythene bag.
 Immediately add 100ul of n-butanol over the gel mix and leave the arrangement for 20 min
undisturbed. Appearance of interface between the top unpolymerized solution and the bottom
separating gel-indicates the completion of polymerization.
 Remove the top Butanol & un polymerized layer by inserting a filter paper and wash briefly
with D. water.
 Prepare the following Stacking Gel mixture (5%)

Component For 1 gel For 2 gel

Acrylamide monomer 0.333ml 0.666ml
4x separating gel buffer (pH 6.8) 0.500ml 1.000ml
10% SDS 0.020ml 0.040ml
10% APS 0.010ml 0.020ml
D. water 1.137ml 2.274ml
TEMED 1-2ul (Final volume 2.0ml) 4ul
  Mix gently, pour on top of the separating buffer gel and insert the comb slowly to 0.8cm
depth. Leave the arrangement for 20 min.
 Seal the bag and keep undisturbed at room temperature.

 Preparation of protein sample:

Key elements:
1. Protein sample
2. 2x Sample Treatment Buffer (Bromophenol blue)

Protocol:
 Take samples in different concentrations in 10ul volume and add 10ul 2x Sample Treatment
Buffer.
 Keep the samples in boiling water (95°C) bath for 3 minutes.

 Gel running:
 Cut open the polythene pouch and take the gel.
 Remove the comb slowly, if there is buffer in the wells remove by filter paper.
 Clamp the sandwich on the tank, the notched alumina plate facing the notch of the tank.
 Apply samples & standard MW marker (protein ladder) to the wells and slowly layer the tank
buffer on to each sample.
 Pour tank buffer 35ml to each Bottom and top buffer reservoir slowly.
 Connect the apparatus to the power pack and run at cc-mode of 10-15 mA till BPB dye
reaches the separating gel. Then at CV mode of 10-150V till the BPB tracking dye reaches the
bottom of the gel.

 Gel staining:
 After the run, take out the gel plate, remove the side spacers and lift the alumina plate using a
spatula.
 Take a gel developing tray and pour about 25ml of stain.
 Slowly tilt the rectangular glass plate and drop the gel in to the stain.
 Gently agitate the gel in stain for 30 minutes (200 rpm).
 Decant the stain and add 25 ml of Destain-I and gently agitate for 30 minutes.
 Decant the Destain-1, and add 25 ml of Destain- II and agitate slowly for 30 minutes till a
clear background appears.
 Take a photograph and analyse the images in a gel doc system. MW determination &
densitometric scanning can be done.