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
- Add 60 g of Sephadex G-50 [Sigma S-5897] to 960 mL of ddH20 and stir for 10 minutes.
- Let solution settle for 10–20 minutes and pour off H20.
- Repeat steps 1–2 three times.
- Add TE [pH 7.6–8.0] to a total volume of 960 mL.
- Random hexamer pd(N)6 5'–PO4 Na+ salt; 1 unit = 26.5 µg.
- Dilute 50 units with 500 µL of TE for stock that is 100 units/mL.
- 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]
- Add 1 g of salmon sperm DNA to 100 mL of 0.4 M NaOH and mix to dissolve.
- Boil for 30 minutes in a water bath, place on ice to cool, then cool to room temperature.
- Adjust pH to 7.0 using glacial acetic acid.
- Aliquot 15 mL to each of seven 50-ml tubes.
- Add two volumes of cold 100% ethanol and store at -20°C for at least 1 hour.
- Centrifuge at 6,000 RPM for 20 minutes.
- Wash pellet with 70% ethanol.
- Dry pellets and combine.
- Resuspend in 100 mL ddH20.
- Final concentration is 10 mg/mL.