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JR4915, huFcRn line 32, will give a transgene band ~740 bp.
JR4919, huFcRn line 276 will give a product ~350 bp (because 32 is based on genomic DNA and 276 on cDNA).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
oIMR7718 | AGC CAA GTC CTC CGT GCT C | Transgene Forward | A | FCGRT | ||
oIMR7719 | CTC AGA GAT GCC AGT GTT CC | Transgene Reverse | A | FCGRT | ||
oIMR8744 | CAA ATG TTG CTT GTC TGG TG | Internal Positive Control Forward | B | |||
oIMR8745 | GTC AGT CGA GTG CAC AGT TT | Internal Positive Control Reverse | B |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTPS-kapa | 0.26 mM |
oIMR7718 | 0.50 uM |
oIMR7719 | 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 |
oIMR8744 | 0.50 uM |
oIMR8745 | 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 |
---|---|
021146 | B6.Cg-Fcgrttm1Dcr Prkdcscid Tg(CAG-FCGRT)276Dcr/DcrJ |
004919 | B6.Cg-Fcgrttm1Dcr Tg(CAG-FCGRT)276Dcr/DcrJ |
014565 | B6.Cg-Fcgrttm1Dcr Tg(FCGRT)32Dcr/DcrJ |
016919 | B6.Cg-Rag1tm1Mom Fcgrttm1Dcr Tg(CAG-FCGRT)276Dcr/DcrJ |
029686 | B6.Cg-Tg(FCGRT)32Dcr Fcgrttm1Dcr Ighg1em2(IGHG1)Mvw/MvwJ |
025099 | BXSB.Cg-Fcgrttm1Dcr Tg(FCGRT)99Dcr/J |
028616 | NOD.Cg-Tg(CAG-FCGRT)276Dcr Fcgrttm1Dcr Prkdcscid Il2rgtm1Wjl/J |
7 strains use this protocol |