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Stock No: 018980
Protocol 32108: Probe Assay - Tg(Ddx4-cre)1Dcas-Chr18-alternate1
Version 1.0
Notes
Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX).
To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility.
Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.
Expected Results
Mutant= 136 bp
Wild Type = 149 bp
This assay can be run as a standard assay. Run separated and omit probes.
This assay is capable of distinguishing hemi from hom. Transgene insertion site is known to be on mouse Chr 18.
This assay is designed around this insertion site, but it has not been tested on hom animals.
This assay is NOT able to be used for copy number evaluation. if this is required, it is suggested to type by qPCR.
Sequence
Wt Sequence: tgggcagtctatccttcagcactaaggtaaaatcatgattaaaatgagtgccccgaaggccattcacttaactcacttggggccaggaaataaaatttgaaaagTGTCACTCTAGCTGGAATTCTATTGAAATGTTTCTAACTTCTCATAATTGAAATACGTAAATTGACCATCTAGGGTAGAGGAGGAACAGCTGAGTCCTGAGAATCTCAGCGAAGGAGCA
Mutant Sequence: CAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCatGTCACTCTAGCTGGAATTCTATTGAAATGTTTCTAACTTCTCATAATTGAAATACGTAAATTGACCATCTAGGGTAGAGGAGG
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 38353 | TTA AAA TGA GTG CCC CGA AG | Wild type Forward | A | mChr18 | ||
| 38354 | CTC CTC TAC CCT AGA TGG TCA A | Common | A | mChr18 | ||
| 38355 | Fluorophore-1 | CAT TCA CTT AAC TCA CTT GGG GC | Quencher-1 | WT Probe | mChr18 | |
| 38356 | TTT TCA CTG CAT TCT AGT TGT GG | Mutant Forward | A | SV40pA | ||
| 38357 | Fluorophore-2 | TTT GTC CAA ACT CAT CAA TGT ATC TTA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 38353 | 0.40 uM |
| 38354 | 0.40 uM |
| 38356 | 0.40 uM |
| Wt Probe | 0.15 uM |
| Mutant Probe | 0.15 uM |
| DNA |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 95.0 | -- | |
| 2 | 95.0 | -- | |
| 3 | 60.0 | -- | |
| 4 | -- | repeat steps 2-3 for 40 cycles | |
| 5 | 40.0 | -- | Forever |
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.