Primers for Unknown Genes

[hsip_15]

I wish to ask if there are any ways to construct the primers for unknown genes in a plasmid?

I looked through some other forums and they have suggested working backwards(from protein mutations and such) or using some random primers?

What does working backwards means?

What does a random primer do to help in this matter?

Any comments and replies are welcomed. Thanks.

[kmunson779]

I'm not sure I completely understand your question.

You have a plasmid that contains some unknown gene(s)?

It seems to me the best way to go would be to start sequencing, from either the backbone sequence if you know what plasmid it is, or from your resistance gene sequence...

Make a primer, sequence your plasmid with it, then design a new primer near the end of your read and sequence with it, etc, etc.

I know there's a way to use degenerate primers based on a protein sequence to attempt to amplify the gene... try looking at the below site for one example. (add the usual web prefix to the beginning; the forum won't let me post links)

bioinformatics.weizmann.ac.il/~pietro/papers/CODEHOP.pdf

If you can specify what exactly you're trying to do, maybe I or someone else can help more.

[oBWhat]

Yeah, the best thing to do is sequence either side (5' and 3') of the multiple cloning site.

[admin]

Great posts oBwhat and kmunson, I also think that the plasmid should be sequenced and good primers selected from the sequence.

Once you sequence the plasmid from the MCS, you can easily do a BLAST and see the identity of the gene and also pick primers.