100 mM calcium chloride (CaCl2):

1.48 g CaCl2-2H₂O

ddH₂O to 100 ml

autoclave to sterilize (store at 4deg.C).

50 mM calcium chloride:

0.74 g CaCl2-2H₂O

ddH₂O to 100 ml

autoclave to sterilize (store at 4deg.C).

Deionized formamide:

Stir formamide (Schwarz/Mann Biotech 800686) with Amberlite MB resin, 10 g. per 100 ml, for one hour to deionize; filter through Whatman 3MM paper, store in a dark bottle at room temperature or 4deg.C.

10X denaturing buffer: 200 mM Tris-HCl, pH 9.5, 1 mM EDTA, 10 mM spermidine in double distilled water.

2 ml 1 M Tris-HCl, pH 9.5

20 ul 0.5 M EDTA, pH 8.0

1 ml 100 mM spermidine

ddH₂O to 10 ml (aliquot and store at -20℃)

Diatomaceous earth (100 mg/ml): Suspend 10 g of diatomaceous earth (Sigma D-5384) in 100 ml of distilled water in a 100 ml graduated cylinder, and let it settle down for 3 hours. Decant the supernatant, and resuspend the pellet in 100 ml of 6 M guanidine HCl, pH 6.4, containing 50 mM Tris-HCl, 20 mM EDTA.

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 -20℃).

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 -20℃).

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