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Wheat Genetics Resource Center

Kansas State University
Department of Plant Pathology

4024 Throckmorton
1712 Claflin Road
Manhattan, KS 66506-5502
785-532-6176 (TEL)
785-532-5692 (FAX)
wgrc@k-state.edu

Wheat Genetics Resource Center
Kansas Wheat Innovation Center

1990 Kimball Ave
Manhattan, KS 66502
785-320-4383 (TEL)

 

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.