Protocol For Genotyping ERKO Mice
|
Oligos: |
UY83 (P1); UV182 (P2); WR115 (P3); WR111 (P4) |
|---|---|
| P1 | 5′ TTG CCG ATA ACA ATA ACA T 3′ |
| P2 | 5′ ATT GTC TCT TTC TGA CAC 3′ |
| P3 | 5′ GGC ATT ACC ACT TCT CCT GGG AGT CT 3′ |
| P4 | 5′ TCG CTT TCC TGA AGA CCT TTC ATA T 3′ |
PCR Mixes (for 10 samples):
| Solution for Primer Mix A | 1X | 11X |
| PCR Supermix | 22.5ml | 247.5ml |
| P1/P2 | 0.5ml /0.5ml | 5.5ml/5.5ml |
| Solution for Primer Mix B | 1X | 11X |
| PCR Supermix | 22.5ml | 247.5ml |
| P3/P4 | 0.5ml /0.5ml | 5.5ml/5.5ml |
- Mix well by vortexing.
- Aliquot 23.5ml of mix A and mix B into designated tubes (20 total= 10A + 10B).
- Add 2μl of DNA to the appropriate A and B aliquots. (Note: There should be an A and B for each DNA sample)
- Mix well by pipetting the sample up and down 3 times leaving no bubbles.
- Add 40ml of mineral oil to the top of the sample in the tube.
PCR Conditions:
- 94oC for 1 min @ File 45 (1) or 43 (2), link to…
- (94oC for 30 sec, 55oC for 30 sec, 72oC for 1 min) X30 @ File 46 (1) or 44 (2), link to…
- 4oC soak @ File 99
Gel Electrophoresis:
- Use 1% agarose gel + 5ml of Ethidium bromide.
- 10ml PCR product + 2ml of loading buffer (6X)
- 7ml of DNA ladder.
- Run at 100 Volts.
Identifying the Genotype:
| HT (-/+) | KO (/) | WT(/) | |
| Primer mix A: | 387bp | 387bp | |
| Primer mix B: | 255bp | 255bp | 815bp |
