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Transgene = ~180 bp
Wt = 349 bp
>chr14:97484687-97485035 349bp TCGTACCCTCATTCCCTCTG TGTAGGGGCAATCCATTTTC This assay can differentiate between Tg/0 & Tg/Tg Integration site is known.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
39284 | TCG TAC CCT CAT TCC CTC TG | Wild type Forward | A | Wt F | ||
39285 | TGT AGG GGC AAT CCA TTT TC | Common | A, B | |||
39286 | ACC GAT AAA ACA CAT GCG TCA | Transgene Forward | B | Tg F |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTPS-kapa | 0.26 mM |
39284 | 0.50 uM |
39285 | 0.50 uM |
Glycerol | 6.50 % |
Dye | 1.00 X |
Kapa 2G HS taq polym | 0.03 U/ul |
DNA |
Step | Temp °C | Time | Note |
---|---|---|---|
1 | 94.0 | -- | |
2 | 94.0 | -- | |
3 | 65.0 | -- | -0.5 C per cycle decrease |
4 | 68.0 | -- | |
5 | -- | repeat steps 2-4 for 10 cycles (Touchdown) | |
6 | 94.0 | -- | |
7 | 60.0 | -- | |
8 | 72.0 | -- | |
9 | -- | repeat steps 6-8 for 28 cycles | |
10 | 72.0 | -- | |
11 | 10.0 | -- | hold |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTPS-kapa | 0.26 mM |
39285 | 0.50 uM |
39286 | 0.50 uM |
Glycerol | 6.50 % |
Dye | 1.00 X |
Kapa 2G HS taq polym | 0.03 U/ul |
DNA |
Step | Temp °C | Time | Note |
---|---|---|---|
1 | 94.0 | -- | |
2 | 94.0 | -- | |
3 | 65.0 | -- | -0.5 C per cycle decrease |
4 | 68.0 | -- | |
5 | -- | repeat steps 2-4 for 10 cycles (Touchdown) | |
6 | 94.0 | -- | |
7 | 60.0 | -- | |
8 | 72.0 | -- | |
9 | -- | repeat steps 6-8 for 28 cycles | |
10 | 72.0 | -- | |
11 | 10.0 | -- | hold |
Stock Number | Strain Name |
---|---|
031010 | STOCK Gt(ROSA)26Sortm1(rtTA,EGFP)Nagy TgTn(pb-tetO-Pou5f1,-Klf4,-Myc,-Sox2,-mCherry)250Nagy Tg(Pou5f1-EGFP)1Nagy/J |
031012 | STOCK Gt(ROSA)26Sortm1(rtTA,EGFP)Nagy TgTn(pb-tetO-Pou5f1,-Klf4,-Myc,-Sox2,-mCherry)250Nagy/J |
031011 | STOCK Gt(ROSA)26Sortm1.1(rtTA,EGFP)Nagy TgTn(pb-tetO-Pou5f1,-Klf4,-Myc,-Sox2,-mCherry)250Nagy Tg(Pou5f1-EGFP)1Nagy/J |
031013 | STOCK Gt(ROSA)26Sortm1.1(rtTA,EGFP)Nagy TgTn(pb-tetO-Pou5f1,-Klf4,-Myc,-Sox2,-mCherry)250Nagy/J |
4 strains use this protocol |