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

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SECOND DAY

Gel

• 1 1% agarose gel per pair
• SYBR Safe 1 uL/10 mL agarose
• small gel (50 mL)
• ? small tooth comb, 2 per gel
• 1X TAE
• loading dye
• DNA standard

BLAST

Use primer sequences?

Analyze Gel

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