Primer Design

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jamesgoat
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Primer Design

Post by jamesgoat » Tue Jun 06, 2006 3:34 am

Hi, I am trying to fish a gene from bacterial genome. I've already tried six or seven pairs of primer and even degenerated primers. Have anyone ever experienced this? Thanks.

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LilKim
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Post by LilKim » Tue Jun 06, 2006 4:59 am

what exactly do you mean by "fishing" a gene .... what exactly have you done so far?

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Post by jamesgoat » Tue Jun 06, 2006 1:38 pm

I am designing primers from the N-terminal amino acid sequences I obtained, taking into consideration of codon usage. What I tried to do is to pull out the full gene from the bacteria genome/pladmid. So far, no luck. Any other approach or suggestion?

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Post by oppox » Wed Jun 07, 2006 7:26 am

Im still alittle confused of what u have done. You have a set of primers that is complementary to the 5´ and 3´ end of the gene and u cant fish it out?
I got a little confused because u talked about codon use, since it doesnt have anything to do with the PCR reaction.
Lower the temperatures may be a way if it contains alot of A and T:s. Are u trying to fish it out from colonies or from isolated plasmids?

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Post by jamesgoat » Wed Jun 07, 2006 2:52 pm

I considered codon usage because I designed the primers based on the amino acid sequences. I just wonder whether the primers are specific for the particular species I am working. I fished from isolated plasmid, cloned the PCR fragments into vector and sequenced them. So far, I didn't get the gene or similar gene from the PCR. Thanks.

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Post by LilKim » Wed Jun 07, 2006 3:01 pm

Have you been able to amplify anything?

I'm assuming that you're using degenerate primers (maybe with Inosine at the "wobble" position for the 5' oligo. and then another degenerate primer at the 3' end)

Umm.. as far as 'fishing out' an entire gene on one plasmid in a library ... Gosh! that's a shot in the dark. It may work... but for most normal people (like myself) the gene is probably to big to fit into a plasmid. (parts could be on 2,3, 4 or 5 plasmids.... ESPECIALLY if you're working witha genomic Vs. cDNA)

Overall it just sounds like a difficult task that will take alot of time and patience. But, i think it can be done (although i've never done it before). You're just really going to have to think-through your experimental plan. (like i'd spend a good week trying to figure it out.) Maybe you could consider using another genetics based aproach to figure out the gene that produces the protien?.

Personally, it would take me some time to give you really sound advice or help you plan your project ... because, i simply don't know enough to specificially direct you. But, if you'd like my opinion please let me know ... i'll be happy to help (although I definitely don't know everything... i'm still a student !)

best wishes!
- KIM

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Primer Design

Post by jamesgoat » Tue Jun 13, 2006 3:30 pm

Thanks for all the responses. I appreciated them. I only use a pair of degenerated primers one time. It didn't work so I switched back to specific primer pairs. Maybe I can describe a little bit about my experimental design here. I found a protein that is differentially expressed in my bacteria under a condition. So I obtain ~ 30 N-terminal amino acids. I blasted against the NCBI database and it resembles an outer membrane protein. From the a.a. , I tried to design primer pairs to pull out the gene either from genomic or plasmid DNA. The PCR did amplify some fragments. I cloned and sequenced them but they were not the protein I found earlier and not the outer membrane proteins either. The database has the full sequence of the gene in the bacteria but I just could not pull out the gene. It's really intriguing....Does any one have other approaches?

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