| Today's date: | 9/19/05 | ||||||||
| PCR name: | JBM | ||||||||
| Purpose: | Distinguish between Irs1 Jnk binding mutant (JBM) knockin and control knockin (CK) alleles. Note: this is an allele-specific PCR; a wt (+) allele, gives same bands as CK allele. Digest is not required. | ||||||||
| Samples: | |||||||||
| 1 | |||||||||
| 2 | |||||||||
| 3 | |||||||||
| 4 | |||||||||
| 5 | |||||||||
| # of rxns | |||||||||
| Reactions (ul): | X | 18 | Profile name: | JBM&S7A | |||||
| DNA | 1 | step | time (min:sec) | temp (C) | |||||
| H20 | 11.5 | 207 | 1 | 15:00 | 95 | ||||
| 10X buffer | 2 | 36 | 2 | :30 | 94 | ||||
| 4mM dNTPs | 1.5 | 27 | 3 | :20 | 64 | ||||
| 10pmol/ul primers | 4 | :45 | 72 | ||||||
| JBDF | 1 | 18 | go to | step 2 | 34 times | ||||
| LLR | 1 | 18 | 5 | 10:00 | 72 | ||||
| GGF | 1 | 18 | 6 | hold | 4 | ||||
| JBDR | 1 | 18 | |||||||
| 20 | ul | 342 | ul | ||||||
| + HotStar Taq | 0.18 | ul | 3.24 | ||||||
| Primers: | Sequence: (5' to3') | Tm | |||||||
| JBDF | GAACATGTCCCCAGTGGGAGATTCC | 61.1 | |||||||
| LLR (RPTRLSL) | CGAACCCGGGGTAAGGTGCTT | 62.0 | |||||||
| GGF (RPTRGSG) | CGACCCACAAGGGGGTCCGG | 65.3 | |||||||
| JBDR | CGAGCCAGTCTCTTCTCTAGGAGCT | 61.0 | |||||||
| Products: | from allele(s) | length (bp) | digests? | primers | |||||
| common outer | all alleles | 612 | Nhe I (JBM)* | JBDF + JBDR | |||||
| wt-specific (LL) | CK, +, etc | 425 | GGF + JBDR | ||||||
| JBM-specific (GG) | JBM | 242 | JBDF + LLR | ||||||
| (* from the JBM allele only, the common band from outer | |||||||||
| primers can be digested after a buffer change with Nhe I) | |||||||||
| Genotypes: | bands | ||||||||
| +/+ | 612 (faint), 425 | ||||||||
| CK/CK | 612 (faint), 425 | ||||||||
| JBM/CK or JBM/+ | 612 (faint), 425, 242 | ||||||||
| JBM/JBM | 612 (faint), 242 | ||||||||