STOCK Tg(KRT14-cre)1Amc/NJU

For strains: J004782

Protocol Name: / Generic Cre
Reaction Components / Reaction Components / Volume(ul)
MilliQ / 35.5
Primer1(25uM) / 0.5
Primer2(25uM) / 0.5
dNTP(dA/T/C/GTP 10mM each) / 1.0
Mg2+(25nM) / 5.0
Mg2+Buffer(10x) / 5.0
Template / 2.0
CRE-f / 5’-att tgc ctg cat tac cgg tc –3’ / 20bp
CRE-r / 5’-atc aac gtt ttc ttt tcg g -3’ / 19bp
Reaction Conditions
([Mg2+]=2mM) / Step / Temp. / Time / Note
1 / 94 / 3 min
2 / 94 / 20sec
3 / 64 / 30sec / -0.5 cper cycle
4 / 72 / 35sec / Goto step 2, 12 cycles
5 / 94 / 20 sec
6 / 58 / 30 sec
7 / 72 / 35 sec / Goto step 5, 25 cycles
8 / 72 / 2 min
9 / 16 / 12hrs
Result / Trangenic allele can be amplified
a fragment of about 350 bp DNA
while wild type allele can not. /
Remark / The primers are better than Jackson Lab provided (oIMR1084, oIMR1085).

For strains: J004782

Protocol Name: / Tg(KRT14-cre)1Amc
Reaction
Components / Reaction Components / Volume(ul)
MilliQ / 35.5
Primer1(25uM) / 0.5
Primer2(25uM) / 0.5
dNTP(dA/T/C/GTP 10mM each) / 1.0
Mg2+(25nM) / 5.0
Mg2+Buffer(10x) / 5.0
Template / 2.0
oIMR3069 / TTC CTC AGG AGT GTC TTC GC / Transgene
oIMR3070 / GTC CAT GTC CTT CCT GAA GC / Transgene
oIMR7338 / CTA GGC CAC AGA ATT GAA AGA TCT / Internal Positive Control
oIMR7339 / GTA GGT GGA AAT TCT AGC ATC ATC C / Internal Positive Control
Reaction Conditions
([Mg2+]=2.0mM) / Step / Temp. / Time / Note
1 / 95 / 5min
2 / 94 / 30sec
3 / 60 / 35sec
4 / 72 / 45sec / Goto step 2, 35 cycles
5 / 72 / 3min
6 / 16 / Hold
Result / Transgene = 494 bp
Internal Positive Control = 324 bp
Separated by gel electrophoresis on a 1.0% agarose gel.