I checked the hot start PCR, altogether with the three restrictions in an agarose gel and it came out like this:
1) ladder
2) cI I
3) cI II
4) cI III
5) positive control
6) negative control
7) double restriction with EcoRI/PstI
8) single restriction with PstI
9) single restriction with EcoRI
The double restriction didn’t come out well, as we can not see the 1kb band corresponding to the cI inverter.
After that I purified the cI inverter 3 PCRs together using the High Pure PCR Product Purification Kit from Roche.
Then I performed a double restriction of it with EcoRI and SpeI for posterior ligation as follows:
iGEM Project name 1
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