Kansas State University

SOLUTIONS FOR DNA PROCEDURES

100X Denhardt's Solution

100X Denhardt's Solution

polyvinylpyrolidone	10 g	Sigma PVP-360
ficol	10 g	Sigma F-4375
bovine serum albumin	10 g	Sigma A-9647
Bring to a final volume of 500 mL with ddH20 and store at 4°C.

Sephadex in TE 

1. Add 60 g of Sephadex G-50 [Sigma S-5897] to 960 mL of ddH₂0 and stir for 10 minutes.
2. Let solution settle for 10–20 minutes and pour off H₂0.
3. Repeat steps 1–2 three times.
4. Add TE [pH 7.6–8.0] to a total volume of 960 mL.
5. Autoclave.

Oligonucleotide

1. Random hexamer pd(N)₆ 5'–PO₄ Na⁺ salt; 1 unit = 26.5 µg.
2. Dilute 50 units with 500 µL of TE for stock that is 100 units/mL.
3. Make a solution of 59.6 ng/µL oligo. Take 4.5 µL of stock and add 195.5 µL ddH₂0 to give 200 µL at 59.6 ng/µL.

Salmon sperm DNA [10 mg/mL] [source USB #78019]

1. Add 1 g of salmon sperm DNA to 100 mL of 0.4 M NaOH and mix to dissolve.
2. Boil for 30 minutes in a water bath, place on ice to cool, then cool to room temperature.
3. Adjust pH to 7.0 using glacial acetic acid.
4. Aliquot 15 mL to each of seven 50-ml tubes.
5. Add two volumes of cold 100% ethanol and store at -20°C for at least 1 hour.
6. Centrifuge at 6,000 RPM for 20 minutes.
7. Wash pellet with 70% ethanol.
8. Dry pellets and combine.
9. Resuspend in 100 mL ddH₂0.
10. Final concentration is 10 mg/mL.