HotSHOT genomic DNA prep
HotSHOT genomic DNA prep kdorfman Tue, 06/21/2022 - 15:00For 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
Alkaline Lysis Reagent
Alkaline Lysis Reagent kdorfman Tue, 06/21/2022 - 15:07Alkaline 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
pH =~12