2 replies to this topic

[tech123]

Hi all:
I am trying to make RNA probe for ISH. I ordered cDNA clone from a company and I realized that the vector they used does not have T7 or T3 or SP3 promoter sequence. So, how can I add the promoter sequence to my vector? Also, I streaked the glycerol stock which I got yesterday but no colonies today, what could have gone wrong?
Vector:pME18S-FL3
Thanks in advance

[prabhubct]

you have to look for restriction site around SV40 promoter and add same restriction sequence in your T7 promoter flanking primer then amplify T7 promoter from available vector with T7 promoter. and clone into desired vector. May be quiet long route.

whats the problem with SV40 promoter. your vector has it.

[phage434]

The T7 promoter is short enough that you can encode it into the 5' end of a primer.  Make two primers, which prime to your vector, amplifying the whole thing.  On the forward primer, put a T7 promoter.  Order phosphoylated or kinase the primers to add 5' phosphate. Amplify with a blunt end enzyme (not Taq),  cut with DpnI to trash the original vector, ligate with quick ligase, and transform.