- Grew up plasmid (pCMV-BluescriptR-MTMR9, 1.14 mg/ml, from MRC)
- PCR hMTMR9 sequence including BamH1 and Xho1 sites.
Primers:
Forward: 5’- t aGG ATC CCC ATG GAG TTT GCG GAG CTG ATT AAG -3’ name=hMTMR9for.
(5.9 mg/ml)
length: 34
Tm: 66ºC
%GC: 50
Reverse: 5’- taCTCGAG TCAGGGACTCTCCTGCATCCCGTC -3’ name=hMTMR9rev. (4.3 mg/ml)
length: 32
Tm: 68ºC
%GC: 59
- After PCR, run product and recover 1.6 Kb band. Ligate into pCR4-Blunt-TOPO
(->pCR4-hMTMR9)
- Send for sequencing
- Subcloned into:
Prey pACT2………..with BamH1 and Xho1 (pACT2-hMTMR9)
Bait pFBT9…………with BamH1 and Sal1 (pFBT9-hMTMR9)
- For Co-IP, Co-IF:
Digest
pFBT9-hMTMR9 with BamH1 and Xho1
pCMV-HA with Bgl II and Xho1
pEGFP-C2 with Bgl II and Sal1