Zebrafish finclip genotyping

Submitted by kdorfman on Wed, 06/21/2023 - 18:56

Copied from 2022:

*DNA Extraction**

  • Extract DNA from fish fin clips with HotSHOT DNA extraction prep

    • 50 uL alkaline lysis reagent per rxn
    • 50 uL Neutralization buffer per rxn
    • Aliquot 1 mL of each per pair

  • Rolf brings 20 fin clips

  • Students work in pairs, doing 4 clips per pair.

  • Master mix recipe

Set up PCR reactions

  • Use Qiagen Taq PCR Master Mix Kit

  • Dilute primers from Rolf's lab for GFP (final 0.4 uM in mix)

  • Each 25 uL reaction contains:
    • 12.5 uL 2X Master mix
    • enough primer to make 0.4 uM
    • 5 uL DNA
    • water to 25 uL
  • Karlstrom recipe for 900 uL Master Mix with primers: (see Master mix calculator)
    • 3.6 uL each 100uM primer
    • 450 uL 2x master mix
    • 446.4 uL water (to final volume 900 uL)
  • Each individual student tests all 4 of the DNA samples
    • 20 uL per rxn
    • aliquot 180 uL per pair

Make enough master mix for 9 reactions per pair

PCR program (finclip in Main menu) (don't know which thermocycler)

Thermocycler 3 in 364: folder BIOBOO: programs HOT92, ZFPCR

Step time (min) temp comment
1. Initial denaturation 2 94
2. denaturation 0.5 94
3. annealing 0.5 55 ~4C below primer Tm
4. extension/elongation 0.5 72 or one min per kb 1
5. go to 2 38 times
6. final extension 5 72
7. hold forever 15 can cut short and put into fridge

SECOND DAY

Gel

BLAST

Use primer sequences?

Analyze Gel

  • check bands in blue light
  • take cell phone photo
  • send to Rolf with documentation

  1. 45 sec is better for mcherry; can use 45 sec to 1 min for GFP. EGFP and mcherry amplicons are ~500 BP ↩︎