- Grew up plasmid (from Kazusa).
- PCR hMTMR2 sequence including Bgl II and Sal I sites.
Primers:
Forward: 5’- t aAG ATC TCC ATG GAG ACG AGC TCG AGC TGC GAG -3’ name=hmtmr2for.
(3.23 mg/ml)
length: 34
Tm: 68ºC
%GC: 56
Reverse: 5’- gc GTC GAC TTA TAC AAC AGT TTG
GAC AGG AGT G-3’ name=hMTMR2rev. (3.35mg/ml)
length: 33
Tm: 64ºC
%GC: 48
- After PCR, run product and recover 1.94 Kb band. Ligate into pCR4-Blunt-Topo
- Send for sequencing
- Subcloned into:
Prey pACT2…………………...with BamH1 and
Xho1 (pACT2-hMTMR2)
Bait pFBT9………………….…with BamH1
and Sal1 (pFBT9-hMTMR2)
- For Co-IP, Co-IF:
Digest pCR-Topo-hMTMR2 with Bgl II and Sal1
pCMV-HA with Bgl II and Xho1
pEGFP-C2 with Bgl II and Sal1