| Today's date: | 9/19/05 | ||||||||
| PCR name: | C (D1,3+) | ||||||||
| Purpose: | Shows only presence of at least one copy of IRS1 tissue specific knockout (TKO1) allele in completely deleted form (D1,3). Cannot distinguish between heterozygous and homozygous. | ||||||||
| Samples: | |||||||||
| 1 | |||||||||
| 2 | |||||||||
| 3 | |||||||||
| 4 | |||||||||
| 5 | |||||||||
| # of rxns | |||||||||
| Reactions (ul): | X | 7 | Profile name: | JBM&S7A | |||||
| DNA | 1 | step | time (min:sec) | temp (C) | |||||
| H20 | 13.5 | 94.5 | 1 | 15:00 | 95 | ||||
| 10X buffer | 2 | 14 | 2 | :30 | 94 | ||||
| 4mM dNTPs | 1.5 | 10.5 | 3 | :20 | 64 | ||||
| 10pmol/ul primers | 4 | :45 | 72 | ||||||
| Nhe3 | 1 | 7 | go to | step 2 | 34 times | ||||
| UTRrev1 | 1 | 7 | 5 | 10:00 | 72 | ||||
| 6 | hold | 4 | |||||||
| 20 | ul | 133 | ul | ||||||
| + HotStar Taq | 0.18 | ul | 1.26 | ul | |||||
| Primers: | Sequence: (5' to3') | Tm | |||||||
| Nhe3 | GCCTCTCACCCAGACACCGACATCCAGCT | 70.1 | |||||||
| UTRrev1 | AGAGAGAAGCCCTTCTGTGGCTGCTCCAAACACA | 67.5 | |||||||
| Products: | from allele(s) | length (bp) | digests? | primers | |||||
| D13 | Irs1D1,3 (comp. del.) | 396 | Nhe3 + UTRrev1 | ||||||
| Genotype: | bands | ||||||||
| +/+ | none | ||||||||
| D13/+ | 396 | ||||||||
| D13/D13 | 396 | ||||||||
| flox/+ | none | ||||||||
| flox/flox | none | ||||||||