Vectors and Bacterial Strains

Use the expression vector and bacterial strain that was used to produce the cDNA library of interest. METHOD 1. On ten LB agar plates, plate out nonrecombinant bacteriophage X so as to obtain semiconfluent lysis of the bacterial lawn (please see Chapter 2, Protocol 1). 2. Prepare imprints of the lysed lawns on nitrocellulose filters as described in Steps 5-12 of Protocol 1, but omitting the treatment with IPTG. 3. Dilute the preparation of antibody that is to be used for screening 1 10 with...

Dialysis Buffer 622

Prepare 6 liters of Dialysis buffer 6-2-2. Store at 4 C. EDTA To prepare EDTA at 0.5 M (pH 8.0) Add 186.1 g of disodium EDTA*2H2O to 800 ml of H2O. Stir vigorously on a magnetic stirrer. Adjust the pH to 8.0 with NaOH (approx. 20 g of NaOH pellets). Dispense into aliquots and sterilize by autoclaving. The disodium salt of EDTA will not go into solution until the pH of the solution is adjusted to approx. 8.0 by the addition of NaOH.

Buffers and Solutions

Ammonium acetate (10 M) (used as an alternative to dialysis, Step 9) A Dialysis buffer 6-1 (used as an alternative to ethanol precipitation, Step 9) O EDTA (Step 1D) O EDTA-may be used as an alternative to ACD when preparing DNA from whole blood note, however, that ACD is superior for preserving high-molecular-weight DNA. Ethanol (used as an alternative to dialysis, Step 9) Phenol, equilibrated with 0.5 M Tris-Cl (pH 8.0) A IMPORTANT The pH of the phenol must be approx. 8.0 to prevent DNA from...

Edta Autoclave 20

To prepare EDTA at 0.5 M (pH 8.0) Add 186.1 g of disodium EDTA*2H2O to 800 ml of H2O. Stir vigorously on a magnetic stirrer. Adjust the pH to 8.0 with NaOH (approx. 20 g of NaOH pellets). Dispense into aliquots and sterilize by autoclaving. The disodium salt of EDTA will not go into solution until the pH of the solution is adjusted to approx. 8.0 by the addition of NaOH. To prepare a 10 (v v) solution Dilute 1 volume of molecular-biology-grade glycerol in 9 volumes of sterile pure H2O....

Dialysis Buffer

50 mM Tris-Cl (pH 8.0) 10 mM EDTA (pH 8.0) Prepare four lots of 4 liters of dialysis solution and store at 4 C. EDTA To prepare EDTA at 0.5 M (pH 8.0) Add 186.1 g of disodium EDTA*2H2O to 800 ml of H2O. Stir vigorously on a magnetic stirrer. Adjust the pH to 8.0 with NaOH (approx. 20 g of NaOH pellets). Dispense into aliquots and sterilize by autoclaving. The disodium salt of EDTA will not go into solution until the pH of the solution is adjusted to approx. 8.0 by the addition of NaOH.

Gel Electrophoresis of DNA and Pulsed Field Agarose

Protocol 1 Agarose Gel Electrophoresis How to pour, load, and run an agarose gel. Protocol 2 Detection of DNA in Agarose Gels Nucleic acids that have been subjected to electrophoresis through agarose gels may be detected by staining and visualized by illumination with 300-nm UV light. Methods for staining and visualization of DNA using either ethidium bromide or SYBR Gold are described here. The most convenient and commonly used method to visualize DNA in agarose gels is staining with the...

Recipes O Edta

To prepare EDTA at 0.5 M pH 8.0 Add 186.1 g of disodium EDTA 2H2O to 800 ml of H2O. Stir vigorously on a magnetic stirrer. Adjust the pH to 8.0 with NaOH approx. 20 g of NaOH pellets . Dispense into aliquots and sterilize by autoclaving. The disodium salt of EDTA will not go into solution until the pH of the solution is adjusted to approx. 8.0 by the addition of NaOH. To prepare a 10 v v solution Dilute 1 volume of molecular-biology-grade glycerol in 9 volumes of sterile pure H2O. Sterilize the...