Designing PCR Primers for cloning - (Apr/12/2013 )

bob1 on Mon Apr 15 20:38:14 2013 said:

Thanks. It hadn't quite clicked that each primer replicates all the way across until it gets to the end of the gene so it would replicate the free ends.

You have all been so helpful.

Okay, so I just used this information to make primers for my actual project.
The right primer is okay.
The left is giving me issues with regards to GC content, its at 68-70%. Is there a way to lower this other than trying to change up the primers I use, my ending sequence is pretty much full of G and C bases. These are the last 36bp of my protein sequence:
CAG GCC GGT GTG TCC GAC CCG TCC GCT CAC CCG TGA

Is there anyway around this? The lowest I've managed to get the GC for the right primer is 66% using only 15 bases. Will this be okay or should I change my reverse primer and add a few bases from outside my coding sequence to even out the GC count. Would doing so cause issues later on?

Is there anything to worry about if I have a lot of hypothetical proteins?

If this is the last 20 or so bp of the gene coding sequence then there isn't much you can do about it, unless you want to include some of the 3' UTR and use a primer further out.

The G/C content can matter if it leads the primers to form hairpins, you can usually get around this with DMSO in the PCR.

bob1 on Wed Apr 17 01:57:03 2013 said:

Thanks for the reply. So there'd be no issue in shifting the primer slightly out to include bases in the 3' UTR?
If so I think I would prefer to do that just to get the GC content down.

This is a theoretical question I'm working on btw, so I won't actually be doing any PCR.

So long as you include the coding sequence, there shouldn't be a problem with using primers that are outside the coding region. However, for the forward you don't want it to be too far from the promoter such that it falls off the sequence before reaching the ATG.

bob1 on Wed Apr 17 20:47:38 2013 said:

Awesome, thanks.
I have a few questions on restriction enzymes. Is there any determining factor on which enzyme you use for the forward and reverse primer? I often see protocols specifying that BamHI for instance be put on the forward primer...is this like a hard ands fast rule? Does it have anything to do with the position of the restriction sites on cloning vectors?

It mostly has to do with the position on the vector relative to the promoter site.

Just wanted to post a thank you to eveyone who replied to this topic and helped me out with a special shout out to bob1 I got an A on my paper and commended on my primer design so thank you so much.