- Grew up plasmid (from Kazusa)
- PCR hMTMR4 sequence including Bgl II and Xho1 sites.
Primers:
Forward: 5’- t aAG ATC TCC ATG GGT GAG GAG GGG CCC CCC A -3’ name=hMTMR4for.
(4.23 mg/ml)
length: 32
Tm: 70ºC
%GC: 63
Reverse: 5’- ta CTC GAG TCA ACT GGA AGC TGT AGC AAT GGG -3’ name=hMTMR4rev. (3.72 mg/ml)
length: 32
Tm: 64ºC
%GC: 50
- After PCR, run product and recover 3.58 Kb band. Ligate into pCR4-Blunt-Topo
- Send for sequencing
- Subcloned into:
Prey pACT2…………………...with BamH1 and
Xho1 (pACT2-hMTMR4)
Bait pFBT9………………….…with BamH1
and Sal1 (pFBT9-hMTMR4)
- For Co-IP, Co-IF:
Digest pCR-Topo-hMTMR4 with Bgl II and Xho1
pCMV-HA with Bgl II and Xho1
pEGFP-C2 with Bgl II and Sal1