- Grew up plasmid (pCMV-Sport6-MTMR8, 1.4 mg/ml, from MRC)
- PCR hMTMR8 sequence including BamH1 and Xho1 sites.
Primers:
Forward: 5’- t aGG ATC CCC ATG GAT CAT ATT ACG GTA CCC AAG -3’ name=hMTMR8for.
(4.7 mg/ml)
length: 34
Tm: 64ºC
%GC: 47
Reverse: 5’- atCTCGAG TCA CTG ATG CTT GGA GTA GTC TGC -3’ name=hMTMR8rev.
(4.3 mg/ml)
length: 32
Tm: 64ºC
%GC: 50
- After PCR, run product and recover 2.1 Kb band. Ligate into pCR4-TOPO (->pCR4-hMTMR8)
- Send for sequencing
- Subcloned into:
Prey pACT2…………………...with BamH1 and
Xho1 (pACT2-hMTMR8)
Bait pFBT9………………….…with BamH1
and Sal1 (pFBT9-hMTMR8)
- For Co-IP, Co-IF:
Digest pFBT9-hMTMR8 with BamH1 and Xho1
pCMV-HA with Bgl II and Xho1
pEGFP-C2 with Bgl II and Sal1