Cloning short sequences - Does PCR can help clones upto 70 bps into a protein sequence (May/08/2007 )
Hello Folks
I got one good question for all you. I am trying to fuse a 21 amino acid sequence at the N-terminus of a protein. The DNA sequence of the protein itself is 2.1 kb. I want to add this 21 amino acid (viz 63 bps) at the N terminus alongwith a restriction site at the beginning, so the reading frame is also conserved. Does PCR can achieve this target. I dont have this 63 bps dNA anywhere, just a sequence. So in anyway, i would have to get it synthesized it as a primer/dNA fragment. The problem is, how i am going to fuse it to my protein sequence.
I will appreciate all your help.
Thanks in advance.
Order sense and antisense of the desired 63 bp sequence and anneal them and digest & ligate it into your vector.
Hi
The problem is that I do not have a proper restriction site, where I want to fuse the sequence. And I want to maintain the reading frame too. Anyway, thanks for suggestion.
Hi
The problem is that I do not have a proper restriction site, where I want to fuse the sequence. And I want to maintain the reading frame too. Anyway, thanks for suggestion.
couldnt you clone this gene into a different vector like pBS or pGEm so that you might have the appropriate sites.
Hi
The problem is that I do not have a proper restriction site, where I want to fuse the sequence. And I want to maintain the reading frame too. Anyway, thanks for suggestion.
couldnt you clone this gene into a different vector like pBS or pGEm so that you might have the appropriate sites.
The problem is not with the vector, I can clone my gene into a set of sequences at both ends. The problem is how should I attach this short sequence in front of my protein. The difficulty is I can ligate the new external fragment at the 5` position to the vector but I need to fuse its 3` with the 5` of the protein which is in turn joined at its 3` to the vector.
Hi
The problem is that I do not have a proper restriction site, where I want to fuse the sequence. And I want to maintain the reading frame too. Anyway, thanks for suggestion.
couldnt you clone this gene into a different vector like pBS or pGEm so that you might have the appropriate sites.
The problem is not with the vector, I can clone my gene into a set of sequences at both ends. The problem is how should I attach this short sequence in front of my protein. The difficulty is I can ligate the new external fragment at the 5` position to the vector but I need to fuse its 3` with the 5` of the protein which is in turn joined at its 3` to the vector.
If you can clone the gene into a vector such as pcDNA or any other where the original vector is for making fusion protiens, you could use the sites so that everything is in frame. So you could design 63bp with sites and digest and clone them. If still not possible, I need to c your vector to help you further.
Ehh...
How about designing a pair of primer targeting the gene, and the 63 bp and RE site are added to the 5'-end of the reverse primer?
How about designing a pair of primer targeting the gene, and the 63 bp and RE site are added to the 5'-end of the reverse primer?
Thats a good suggestion. Has any body else tried that, I doubt that primers with such a long over hang can bind to the target sequence. Do you have any experimental evidence. Thanks.
Hi makosad05,
Sorry for late reply.
I personally don't have the experience of using such a long overhang primer. However, a friend of mine had used a primer with 63 bp overhang (yours one should be longer, 70+ bp). She told me that the PCR reaction was more difficult and required a better DNA polymerase (she used the one from Roche).
Hope that this help. Good luck..
I have often used primers with overhangs up to 75 bps long (total primer size 100bp). I use KODhifi and have not exprienced any problems. The tm of the template binding section of said primers are often set at 58 Celsius.