4 replies to this topic

[Curtis]

Hi,

I need to add my gene of interest between T7 promoter and T7 terminator, but pcDNA3 and many other vectors I checked don't have the terminator. So how?

[renee]

Curtis, on 08 June 2011 - 05:01 AM, said:

Hi,

I need to add my gene of interest between T7 promoter and T7 terminator, but pcDNA3 and many other vectors I checked don't have the terminator. So how?

I normally design a primer with the T7 promoter sequence in the 5' end and the T7 terminator at the 3' end with 10-20 bp overlap of your gene of interest or vector. I will attach a document from Thermo Scientific that might help.

Attached Files

•  T7-IRES-Kozak Protocol.pdf   188.34K   262 downloads

[zincfinger]

renee, on 08 June 2011 - 07:21 AM, said:

Curtis, on 08 June 2011 - 05:01 AM, said:

Hi,

I need to add my gene of interest between T7 promoter and T7 terminator, but pcDNA3 and many other vectors I checked don't have the terminator. So how?

I normally design a primer with the T7 promoter sequence in the 5' end and the T7 terminator at the 3' end with 10-20 bp overlap of your gene of interest or vector. I will attach a document from Thermo Scientific that might help.

Hi,

I had the same problem. Have you tried pTNT vector from Promega? It has got both T7 promoter and T7 terminator sequences and very efficient for translations.

[Curtis]

renee, on 08 June 2011 - 07:21 AM, said:

Curtis, on 08 June 2011 - 05:01 AM, said:

Hi,

I need to add my gene of interest between T7 promoter and T7 terminator, but pcDNA3 and many other vectors I checked don't have the terminator. So how?

I normally design a primer with the T7 promoter sequence in the 5' end and the T7 terminator at the 3' end with 10-20 bp overlap of your gene of interest or vector. I will attach a document from Thermo Scientific that might help.

Thank you so much, That's what I thought too.

I got another problem too: I need to add an HDV ribozyme gene just before the terminator and I don't know how to do that. I checked NCBI those genes are 85 bp. I think I need to ask someone to give that to me.

[Curtis]

zincfinger, on 08 June 2011 - 07:23 AM, said:

Hi,

I had the same problem. Have you tried pTNT vector from Promega? It has got both T7 promoter and T7 terminator sequences and very efficient for translations.

thanks, I found it

http://www.promega.c...9/3602MAw4.ashx

this is good.what a big terminator though. it has a small size too. my full-length gene is 15k