Hot Shot DNA PCR Assay

“HotSHOT” genomic DNA preparation

(Hot sodium hydroxide and tris)
From Biotechniques. 2000 Jul;29(1):52,54

ALKALINE LYSIS REAGENT

Reagent [Final] Add Of
NaOH 25mM 125λ 10N NaOH
EDTA 0.2mM 20λ 0.5M EDTA
50ml ddH2O

pH will be 12
EDTA = disodium EDTA

NEUTRALIZATION BUFFER

Reagent [Final] Add Of
Tris-HCl 40mM 325mg Tris-HCl
50ml ddH2O

pH will be 5

 

PROTOCOL

  1. Obtain tissue
    1. 0.2 cm tail snip
    2. 2 mm ear biopsy
  2. Place tissue in 96-well plate
  3. Add 75λ of Alkaline Lysis Reagent
  4. Heat to 95℃ for 10 min to 1 hr (30 min is optimal)
  5. Cool to 4℃
  6. Add 75λ Neutralization Buffer
  7. Use 1 to 5λ per PCR reaction

 

NOTES

  • DNA is suitable for PCR reactions but NOT for Southerns
  • Heating for longer than 30 minutes does not increase [DNA]
  • pH of reagents does not need to be altered
  • Do not worry about undigested floating tissue
  • DNA yield is similar for tail snips and ear punches
  • Too much tissue will destroy PCT attempts
  • DNA must be stored at 4C or -20C

 

From Mike Charles 10/15/03
mikchar@umich.edu
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