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    Alkaline Lysis Buffer A Recipe

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    me_dayakar
    The document provides recipes and protocols for common laboratory solutions and procedures including alkaline lysis buffers, carmalum stain, Coomassie blue solution, LB agar, SSC, DNA loading buffer, TAE buffer, TBE buffer, TE buffer, Tris glycine buffer, X-gal staining solution, E. coli competent cell preparation, and phenol purification of DNA from low melting point agarose. The recipes and protocols describe the ingredients, concentrations, and steps to make these common laboratory reagents and perform standard molecular biology techniques.

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    Attribution Non-Commercial (BY-NC)
    Download as DOCX, PDF, TXT or read online from Scribd

    Alkaline Lysis Buffer A Recipe

    Uploaded by

    me_dayakar
    219 views5 pages
    The document provides recipes and protocols for common laboratory solutions and procedures including alkaline lysis buffers, carmalum stain, Coomassie blue solution, LB agar, SSC, DNA loading buffer, TAE buffer, TBE buffer, TE buffer, Tris glycine buffer, X-gal staining solution, E. coli competent cell preparation, and phenol purification of DNA from low melting point agarose. The recipes and protocols describe the ingredients, concentrations, and steps to make these common laboratory reagents and perform standard molecular biology techniques.

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    Protocol Book

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    The document provides recipes and protocols for common laboratory solutions and procedures including alkaline lysis buffers, carmalum stain, Coomassie blue solution, LB agar, SSC, DNA loading buffer, TAE buffer, TBE buffer, TE buffer, Tris glycine buffer, X-gal staining solution, E. coli competent cell preparation, and phenol purification of DNA from low melting point agarose. The recipes and protocols describe the ingredients, concentrations, and steps to make these common laboratory reagents and perform standard molecular biology techniques.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as DOCX, PDF, TXT or read online from Scribd
    Download as docx, pdf, or txt
    219 views5 pages

    Alkaline Lysis Buffer A Recipe

    Uploaded by

    me_dayakar
    The document provides recipes and protocols for common laboratory solutions and procedures including alkaline lysis buffers, carmalum stain, Coomassie blue solution, LB agar, SSC, DNA loading buffer, TAE buffer, TBE buffer, TE buffer, Tris glycine buffer, X-gal staining solution, E. coli competent cell preparation, and phenol purification of DNA from low melting point agarose. The recipes and protocols describe the ingredients, concentrations, and steps to make these common laboratory reagents and perform standard molecular biology techniques.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as DOCX, PDF, TXT or read online from Scribd
    Download as docx, pdf, or txt
    of 5

    Alkaline Lysis Buffer A Recipe

    1. Add the following to 900ml distilled H2O


     9g Glucose
     3g Tris
     20ml of 0.25M EDTA
    2. q.s. to 1000ml with distilled H2O
    3. Autoclave using standard conditions
    4. Allow to cool to room temperature
    5. Store at 4oC

    When ready to use add 0.02g lysozyme to 100ml of alkaline lysis buffer A to yield alkaline lysis
    buffer 1.

    Alkaline Lysis Buffer 2 Recipe


    1. Add the following to 100ml distilled H2O
     5ml of 20% SDS
     2ml of 10M NaOH

    Alkaline Lysis Buffer 3 Recipe


    1. Add the following to 100ml distilled H2O
     29.4g KAc
     11.5ml of Glacial Acetic Acid

    Carmalum Stain Recipe

    Stock solution

    1. Add 10g of carmalum to 200ml of distilled water


    2. Slowly add in 5ml of glacial acetic acid
    3. Boil for approximately 1 hour
    4. Allow to cool
    5. Filter
    Carmalum stain is used for cell nuclei.

    Cautions: ALWAYS wear personal protective equipment when in the laboratory with
    concentrated acids (or any other time)! Always add acid slowly into water and do not add water
    into acid!

    Coomassie Blue Solution Recipe


    1. Dissolve 2g Coomassie Blue (Serva Blau) in 250ml water
    2. Slowly add 75ml of glacial acetic acid
    3. Add 500ml of ethanol
    4. q.s. to 1000ml with water

    Final concentrations:
    0.2% Coomassie Blue
    7.5% Acetic Acid
    50% Ethanol

    LB Agar Recipe

    1. Add the following to 800ml H2O


     10g Bacto-tryptone
     5g yeast extract
     10g NaCl
    2. Adjust pH to 7.5 with NaOH
    3. Add 15g agar
    4. Melt agar into solution in the microwave
    5. Adjust volume to 1L with dH2O
    6. Sterilize by autoclaving

    20X SSC Recipe


    1. Dissolve the following in 800ml of distilled H2O.
     175.3g of NaCl
     88.2g of sodium citrate
    2. Adjust the pH to 7.0 with a few drops of 1M HCl.
    3. Adjust the volume to 1L with additional distilled H2O.
    4. Sterilize by autoclaving.
    10X Xilene Cyanol/Bromophenol Blue DNA loading buffer
    Recipe

    1. Dissolve in 6.25 ml of H2O


    o .025g of Xilene cyanol
    o .025g of Bromophenol Blue
    o 1.25ml of 10% SDS
    o 12.5ml of glycerol

    Note: Sucrose (40%), Ficoll (15%), or glycerol (30%) can be used in place of each other in DNA
    loading buffer

    50X TAE Recipe


    1. Add the following to 900ml distilled H2O
     242g Tris base
     57.1ml Glacial Acetic Acid
     18.6 g EDTA
    2. Adjust volume to 1L with additional distilled H2O

    10X TBE Recipe


    Add the following to 800ml H2O

     108g Tris base.


     55g Boric acid
     9.3g EDTA
    2. Adjust volume to 1L with additional distilled dH2O

    1X TE Recipe
    3. Add the following to 990ml distilled H2O
     10ml  1M Tris-HCl (pH 8.0)
     400µl  0.25 M EDTA
    5X Tris Glycine Buffer Recipe
    1. Dissolve in 700 ml of H2O:
     15.1g Tris base
     94g glycine
     50ml of 10% SDS
    2. After solid is dissolved, adjust volume to 1L with H2O

    X-gal staining solution Recipe

    To 495ml of PBS add

    1. 1.050g Ferrocyanide
    2. 0.825g Ferricyanide
    3. 0.050g NaDeoxycholate
    4. 0.2g MgCl2
    5. 500ul Nonidet-P40
    6. 0.5g X-gal in 5ml N,N-Dimethylformamide

    Protocol for E. coli Competent Cell


    Preparation
    1. Inoculate 5ml of L-broth with a single colony of selected E. coli strain and incubate
    overnight at 37oC. with moderate agitation(~250rpm)
    2. Inoculate 50ml of L-broth with ~100-300ml of the overnight culture and incubate at
    37oC. with moderate agitiation(~250rpm) until the A595=0.375 ( initial inoculum should
    have A595<0.1)
    3. Place culture on ice in autoclaved, prechilled Corex tubes for 10 minutes
    4. Centrifuge cells 7 minutes at 2500rpm(+4oC) and then resuspend pellet (very gently by
    hand-do not vortex) in 10ml CaCl2 solution( 60mM CaCl2, 15% glycerol, 10mM PIPES,
    pH 7.0)
    5. Centrifuge cells 5 minutes at 2500rpm(+4oC) and then resuspend pellet (very gently by
    hand-do not vortex) in 10ml CaCl2 solution
    6. Leave cells on ice for 30 minutes and then centrifuge for 5 minutes at 2500rpm(+4oC)
    and resuspend cells (very gently by hand-do not vortex)in 2ml CaCl2 solution
    7. Aliquot final preparation in 100ml aliquots on dry ice and store at -70oC. (cells should be
    viable for up to a year at -70oC)

    L-broth: 1% bactotryptone, 1% NaCl, 0.5% yeast extract


    Phenol Purification of DNA From Low Melting Point Agarose

    1. Place phenol mix at 37oC for at least 1 hour


    2. Melt excised agarose fragment at 65oC. for 10 minutes or until agarose is molten, then
    place molten agarose at 37oC for 2 minutes
    3. Add 0.5X volume of phenol mix and shake vigorously for 45 seconds
    4. Centrifuge for 5 minutes at room temperature and transfer aqueous layer to clean tube
    5. Repeat phenol extraction and centrifugation step
    6. Transfer aqueous layer to a clean tube and add 1/50 volume of 5M NaCl, shake for 45
    seconds, and centrifuge for 5 minutes at room temperature
    7. Transfer aqueous material to a clean tube, add 2X volume of EtOH and precipitate (either
    15 minutes at -70oC. or at least 1 hour at -20oC)
    8. Centrifuge for 15 minutes and aspirate off aqueous material
    9. Wash pellet in 1X volume of 70% EtOH and repeat centrifugation
    10. Aspirate off aqueous material and air dry pellet for 5 minutes (or dry in speedvac for ~2
    minutes)
    11. Resuspend pellet in 20µl TE buffer

    Phenol mix: 25ml pure phenol, 15ml H2O, 400ml 1M Tris-HCl, pH to 8.0 with 2 drops 5M NaCl

    The usual stock solution concentration of EtBr is 10 mg/ml, or 1000x higher. Are you sure you
    need the lower concentration?
    I would make this by first making a 10 mg/ml stock and diluting 1000x (assuming that this is
    what you really need).

    -phage434-

    By default,
    EtBr is actually 10mg per ml
    we used to convert it to .5 microgram per ml

    -Mazhar Hussain-

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