adding restriction sites to degenerate primers - (Jul/07/2006 )
I have been pursuing a rather elusive bacterial gene for a while now. I finally got success using degenerate primers, but for cloning this gene I would like to add restriction sites to its ends. Is it ok to add restriction sites to 5' end of degenerate primers. Also can these degenerate primers be used for direct sequencing of PCR fragment. I mean is it even worth a try.
Why don't you clone the product of PCR you have already in a vector T and use the restriction sites of the vector??
I agree with aztecan princess.
U will have the advantage of using the sites in the T vector and also u can sequence the insert using T7 or SP6 promoter primers.
Why don't you clone the product of PCR you have already in a vector T and use the restriction sites of the vector??
I agree with aztecan princess.
U will have the advantage of using the sites in the T vector and also u can sequence the insert using T7 or SP6 promoter primers.
Thanks friends, I thought of this too, but my goal is to clone this fragment into a bacillus integration vector and hope for a homology based recombination , this integration vector does not bear the same sites as pGEM or TOPO vector in the correct order for directional cloning. I know I could still clone it in one of the TA vectors get the exact sequence and then amplify it with specific primers with desired restriction sites at the 5' ends.
I guess I was just being lazy, trying to avoid another cloning step ie if I could sequence the fragment directly, add RE sites to my degenerate primers and go directly to cloning it in my integration vector.
[quote name='nifT' date='Jul 10 2006, 12:19 PM' post='58962']
[quote name='scolix' post='58802' date='Jul 8 2006, 01:42 PM']
if you design new primer, you have to standardized any way, I think it's almost the same work. Any way, good look!!!! 
You should have not problem adding restiction sites to the end of the primer. You will probably want to still clone your product into a ta vector in order to grow enough to liagte into your expression vector. I fyou want to clone the product directly, remember to add a couple extra bases after the enzyme site as most enzymes have trouble cleaving near the ends of linear DNA fragments.
I feel you'd better redesign primer with RE site for your cloned fragment generated from degenerate primers, it will be easier...
I agree with others. Its cheap to get new primers and simpler too for further cloning.
personally i would prefer primers with restriction sites due to my very bad experience with t/a cloning...it just did not work...so, use primers with restrictions sites, and design your primers keeping in mind not to use common enzyme sites which could be present in your fragment, and ensure that the addition of sites does not spring the possibility of too many secondary structures forming due to the added sequence.
Thanks a bunch friends!
I did TA cloning in pGem for sequencing. I also sequenced the PCR product directly with degenrate primers, just for the heck of it and ofcourse for future reference. I got the same sequence both ways.
Then I tried two new sets of primers, one with RE sites added to the degenerate ones and another with RE sites added to primers derived from my sequencing results, again just for the heck of it. Both work like a charm! YAYYYY!!
Half my project relies on this construct!
Thanks so much guys!