- Grow up plasmid (pEFBos-3PAP, ~8 Kb, 1mg/ml, from Nandurkar's lab).
- PCR MTMR12 sequence including BamH I and Xho I sites.
Primers:
Forward: 5’- t aGG ATC CCC ATG CTG GGG AAA GGA GTA GTC-3’ name=MTMR12for.
(4.6 mg/ml)
length: 31
Tm: 66ºC
%GC: 55
Reverse: 5’- gaCTCGAG TCACACATCCCCTAGGTCC -3’ name=MTMR12rev. (5.3
mg/ml)
length: 27
Tm: 64ºC
%GC: 59
- After PCR, run product and recover 2.24 Kb band. Ligate into pCR4-Blunt-TOPO (->pCR4-hMTMR12)
- Send for sequencing
- Subcloned into:
Prey pACT2…….with BamH1 and Xho1 (pACT2-hMTMR12)
Bait pFBT9………with BamH1 and Sal1 (pFBT9-hMTMR12)
- For Co-IP, Co-IF:
Digest
pACT2-hMTMR12 with BamH1 and Xho1
pCMV-HA with Bgl II and Xho1
pEGFP-C2 with Bgl II and Sal1