hMTMR5-sbf1 (from Cleary, must be NM-002972):

- Grew up plasmid pMSCVneo-hMTMR5 (Amp)

- PCR hMTMR5 from pMSCVneo-hMTMR5 incorporating EcoR1 and Sal1 sites

- Primers:
Forward for pACT2: 5’- att aGA ATT CCC atg gcg cgg ctc gcg gac tac ttc gtg ct-3’ name=hMTMR5for. (4.2mg/ml)

length: 41
Tm: 72ºC
%GC: 56

Forward for pFBT9: 5’- ta GAA TTC atg gcg cgg ctc gcg gac tac ttc gtg ct-3’ name=hMTMR5for2. (3.7mg/ml)

length: 37
Tm: 70ºC
%GC: 57

Reverse (for both): 5’- tat GTC GAC tca ggc gtc cga cag gca gct ctg ga –3’ name=hMTMR5rev. (5.6mg/ml)

length: 35
Tm: 70ºC
%GC: 60

- After PCR, run product and recover 5.6 Kb band. Ligate into pCR4-TOPO (->pCR4-hMTMR5 for pACT2 and for pFBT9)

- Send for sequencing

- Subcloned into:
Prey pACT2…………………...with Eco and Xho1 (pACT2-hMTMR5)
Bait pFBT9………………….…with Eco and Sal1 (pFBT9-hMTMR5)

- For Co-IP, Co-IF:
Digest pFBT9-hMTMR5 with EcoR1 and Sal1
pCMV-HA with EcoR1 and Sal1
pEGFP-C2 with EcoR1 and Sal1