10X PCR buffer: 500 mM KCl, 100 mM Tris-HCl, pH 8.5, 15 mM MgCl2 in sterile double distilled water

5 ml 1 M KCl

1 ml 1 M Tris-HCl, pH 8.5

150 ul 1 M MgCl2

ddH₂O to 10 ml

PCR Deoxynucleotide Preparation: To make 12.5 ml of the PCR nucleotides at a concentration of 2 mM each nucleotide, combine the following:

250 ul 100 mM dATP

250 ul 100 mM dCTP

250 ul 100 mM dGTP

250 ul 100 mM dTTP

11.5 ml ddH₂O

Aliquot this into 25 tubes with 500 ul in each tube. This will keep the nucleotides from being frozed and thawed too much.

To order these nucleotides, call Pharmacia at 1 800-526-3593. Order the dNTP set: 27-2035-01 dNTP set (100mM each dATP, dCTP, dGTP and dTTP- each in 250 ul volume)$174.00 for the set.

20% PEG/2.5 M NaCl:

7.3 g NaCl

10 g PEG (MW=8000) (Fisher P156-3)

Dissolve in 40 ml double distilled water by stirring, and then adjust the volume to 50 ml.

50% PEG/0.5 M NaCl:

5.85 g NaCl

100 g PEG (MW=8000) (Fisher P156-3)

Dissolve in 100 ml double distilled water by stirring, and then adjust the volume to 200 ml.

PEG:TE rinse solution: 1:3 solution of 20% PEG containing 2.5M NaCl and 10 mM Tris-HCl, pH 8.0 containing 1 mM EDTA in double distilled water.

250 ul 1 M Tris-HCl, pH 8.0

50 ul 0.5 M EDTA

12.5 ml 20% PEG/2.5 M NaCl.

ddH₂O to 37.5 ml

Phenol, TE-saturated: add an equal volume of 10 mM Tris-HCl, pH 7.5-8.0, 1 mM Na2EDTA to ultrapure phenol, mix well, allow phases to separate, remove and discard upper (aqueous) phase. Repeat until the pH of the aqueous phase is between 7.5-8.0 (store at 4℃).

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