Protocol 21905 - Generic Puromycin-QPCR

Notes

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

Sequence

ATGACCGAGTACAAGCCCACGGTGCGCCTCGCCACCCGCGACGACGTCCCCAGGGCCGTACGCACCCTCGCCGC

CGCGTTCGCCGACTACCCCGCCACGCGCCACACCGTCGATCCGGACCGCCACATCGAGCGGGTCACCGAGCTGC

AAGAACTCTTCCTCACGCGCGTCGGGCTCGACATCGGCAAGGTGTGGGTCGCGGACGACGGCGCCGCGGTGGCG

GTCTGGACCACGCCGGAGAGCGTCGAAGCGGGGGCGGTGTTCGCCGAGATCGGCCCGCGCATGGCCGAGTTGAG

CGGTTCCCGGCTGGCCGCGCAGCAACAGATGGAAGGCCTCCTGGCGCCGCACCGGCCCAAGGAGCCCGCGTGGT

TCCTGGCCACCGTCGGCGTCTCGCCCGACCACCAGGGCAAGGGTCTGGGCAGCGCCGTCGTGCTCCCCGGAGTG

GAGGCGGCCGAGCGCGCCGGGGTGCCCGCCTTCCTGGAGACCTCCGCGCCCCGCAACCTCCCCTTCTACGAGCG

GCTCGGCTTCACCGTCACCGCCGACGTCGAGGTGCCCGAAGGACCGCGCACCTGGTGCATGACCCGCAAGCCCG

GTGCCTGA

JAX Protocol

Protocol Primers

Primer	5' Label	Sequence 5' → 3'	3' Label	Primer Type	Reaction
13926		TGC AAG AAC TCT TCC TCA CG		Forward	B
13927		CGA TCT CGG CGA ACA CC		Reverse	B
13928	Fluorophore-1	ACA TCG GCA AGG TGT GGG TCG	Quencher-1
oIMR1544		CAC GTG GGC TCC AGC ATT		Internal Positive Control Forward	A
oIMR3580		TCA CCA GTC ATT TCT GCC TTT G		Internal Positive Control Reverse	A
TmoIMR0105	Fluorophore-2	CCA ATG GTC GGG CAC TGC TCA A	Quencher-2

Reaction A

Component	Final Concentration
Kapa Probe Fast QPCR	1.00 X
ddH2O
oIMR1544	0.40 uM
oIMR3580	0.40 uM
Primer 3	0.40 uM
Primer 4	0.40 uM
Tg Probe	0.15 uM
IC Probe	0.15 uM
DNA

Cycling

Step	Temp °C	Time	Note
1	95.0	--
2	95.0	--
3	60.0	--	repeat steps 2-3 for 40 cycles

JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

Reaction B

Component	Final Concentration
ddH2O
Kapa Probe Fast QPCR	1.00 X
13926	0.40 uM
13927	0.40 uM
Primer 3	0.40 uM
Primer 4	0.40 uM
Tg Probe	0.15 uM
IC Probe	0.15 uM
DNA

Cycling

Step	Temp °C	Time	Note
1	95.0	--
2	95.0	--
3	60.0	--	repeat steps 2-3 for 40 cycles

JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

Strains Using This Protocol

Stock Number	Strain Name
024906	129-Dp(16Tiam1-Il10rb)8Yey/J
039525	B6.129(Cg)-Shank3^em1Yhj/J
040296	B6.Cg-Gt(ROSA)26Sor^{em2(CAG-LOC4337827)Mbu} Ikzf1^em1Mbu Ikzf3^em1Mbu*/J
040297	B6.Cg-Gt(ROSA)26Sor^{em2(CAG-LOC4337827)Mbu} Ikzf1^em1Mbu*/J
040298	B6.Cg-Gt(ROSA)26Sor^{em2(CAG-LOC4337827)Mbu} Ikzf3^em1Mbu*/J
040295	B6.Cg-Gt(ROSA)26Sor^{em2(CAG-LOC4337827)Mbu} Tcf3^em1Mbu*/J
040300	B6.Cg-Gt(ROSA)26Sor^{em2(CAG-LOC4337827)Mbu} Tcf4^em1Mbu*/J
035506	B6.Cg-Gt(ROSA)26Sor^{tm3(CEBPB)Myz}/J
040293	B6.Cg-Pax5^em1Mbu Gt(ROSA)26Sor^{em2(CAG-LOC4337827)Mbu}*/J
038621	B6;129P2-Ctcf^tm2.2Gal/J
039661	B6;129P2-Gt(ROSA)26Sor^{em2(CAG-LOC4337827)Mbu}*/J
003208	STOCK Dnmt1^tm3Jae Hprt1^b-m3 Tg(pPWL512hyg)1Ems/J
040294	STOCK Gt(ROSA)26Sor^{em2(CAG-LOC4337827)Mbu} Ebf1^em1Mbu*/J
040299	STOCK Gt(ROSA)26Sor^{em2(CAG-LOC4337827)Mbu} Prdm1^em2Mbu*/J
031010	STOCK Gt(ROSA)26Sor^{tm1(rtTA,EGFP)Nagy} TgTn(pb-tetO-Pou5f1,-Klf4,-Myc,-Sox2,-mCherry)250Nagy Tg(Pou5f1-EGFP)1Nagy/J
15 strains use this protocol