HotSHOT genomic DNA prep

HotSHOT genomic DNA prep kdorfman Tue, 06/21/2022 - 15:00

For fin clip, tail snip, or ear punch

  • Obtain tissue 2 mm
    • tail snip
    • ear punch
    • fin clip
  • Place tissue in 96 well plate
  • Add 75 uL Alkaline Lysis Reagent for mouse, 50 uL for fish
  • Heat to 95C 30 min (10 min to 1 hr)
  • Cool to 4C
  • Add 75 uL Neutralization Buffer for mouse, 50 uL for fish
  • Use 3 uL (1-5) pre PCR reaction

Notes:

  • DNA is suitable for PCR, not Southerns
  • Heating longer than 30 min does not increase [DNA]
  • pH of reagents does not need to be altered
  • DNA yield is similar for tail snips and ear punches
  • Too much tissue destroys PCR
  • Store DNA at 4C or 20C
HotSHOT DNA protocol handout

Alkaline Lysis Reagent

Alkaline Lysis Reagent kdorfman Tue, 06/21/2022 - 15:07

Alkaline Lysis Reagent for HotSHOT DNA prep

Recipe from Mike Charles 10/15/03; mikchar@umich.edu; retrieved from Craig Albertson's lab, recalculated for1 M NaOH - 10 M will damage your plastic tube!!

Reagent stock conc final conc amt
NaOH 1 M 25 mM 1.25 mL
EDTA 0.5 M 0.2 mM 20 uL
dI water to final volume 50 mL

Calculator

To make mL Alkaline Lysis Reagent
add: mL 1M NaOH
add: uL 0.5M EDTA
plus to final volume: mL H2O

pH =~12

Neutralization Buffer

Neutralization Buffer kdorfman Tue, 06/21/2022 - 15:09

Neutralization Buffer for HotSHOT DNA prep

Dilute stock Tris-HCl to 40 mM

pH =~5