BioProtocols--msol3

Solutions for molecular cloning--1 ) 2 ) 3 ) 4 ) 5 ) 6 ) 7 ) 8 ) 9 )

Diatomaceous earth-wash buffer: 10 mM Tris-HCl,pH 8.0, 1 mM EDTA, pH 8.0, 50% ethanol in double distilled water.

10 ml 1 M Tris-HCl, pH 8.0

2 ml 0.5 M EDTA, pH 8.0

500 ml 100% ethanol (McCormick Distilling Co., Inc.)

ddH2O to 1 L

1 M DTT (Dithiothreitol, Cleland's reagent):

1.54 g DTT (Calbiochem 233155)

ddH2O to 10 ml (aliquot and store at -20deg.C).

DNase-free RNase A: 20 mg/ml RNase A in 1 mM NaOAc, pH 4.5.

200 mg RNase A (Sigma R-5500)

3.3 ul 3 M NaOAc, pH 4.5

ddH2O to 10 ml

boil for 10 minutes (aliquot and store at -20deg.C).

0.5 M EDTA, pH 8.0 (disodium ethylenediamine tetraacetate):

186.1 g Na2EDTA

Dissolve in approx. 400 ml ddH2O, adjust pH to 8.0 with 10 N NaOH, and adjust to 1 liter final volume with distilled water.

100 mM EDTA:

20 ml 0.5 M EDTA

80 ml ddH2O

100 ml

25mM EDTA, 50 mg/ml Blue Dextran - ABI377 Loading Dye/Formamide mixture: Add 0.93g of EDTA to 90 ml water. Then, adjust the pH to 8.0. Bringthe final volume to 100 ml. Next, add 50 mg Blue Dextran to a 1 ml EDTA solution. Add 1 ul of a 1:5 solution of this loading dye:deionized formamide to each sample well for loading onto the ABI377

95% ethanol/0.12 M NaOAc (ethanol/acetate):

95 ml 100% ethanol

4 ml 3 M NaOAc pH 4.5

1 ml ddH2O

100 ml

5 mg/ml ethidium bromide (EtBr):

500 mg EtBr (Sigma E-8751)

ddH2O to 100 ml

10X Freezer Media (FM) for storing either shotgun plasmid-based sub-clone or cDNA clones in microtiter plates):

Final Concentration 1L 500 ml 250 ml

------------------- ------- -------- -------

360 mM K2HPO4 62.7 gm 31.35 gm 15.68 gm

132

mM KH2PO4 17.96 gm 8.98 gm 4.49

gm

17 mM Sodium Citrate 5.0 gm 2.5 gm 1.25 gm

4 mM MgSO4.7H2O 0.98 gm 0.49 gm 0.24 gm

(or 1M MgSO4 4 ml 2 ml 1 ml)

68 mM (NH4)2SO4 8.98 gm 4.49 gm 2.25

gm

44% Glycerol 440

ml 220 ml 110 ml

Bring to volume with dH2O

Sterilize by filtration throught 0.2um filter

Then the final growth and storage media is prepared in a ratio of 9 volumes LB media and 1 volume of this 10X Freezer Media (FM).

FE (formamide/EDTA): 5:1 (v/v) formamide:50 mM EDTA

10 ul ddH2O

10 ul 100 mM EDTA

100 ul deionized formamide

make fresh

10X Fill-in/Kinase buffer: 500 mM Tris-HCl, pH 7.6, 100 mM MgCl2, 10 mM DTT, and 50 ug/ml BSA in double distilled water.

5 ml 1 M Tris-HCl, pH 7.6

1 ml 1 M MgCl2

100 ul 1 M DTT

500 ul 1 mg/ml BSA

3.4 ml ddH2O

10 ml

Commonly used solutions (1)--- 1 ) 2 ) 3 ) 4 ) 5 ) 6 )

Commonly used solutions (2)--- 1 ) 2 ) 3 )

Solutions for molecular cloning---1 ) 2 ) 3 ) 4 ) 5 ) 6 ) 7 ) 8 ) 9 )