PCR Primer again - (Jan/30/2007 )

Hi !!!

I am having trouble desiging primers with restriction sites. I need to use them with a pET vector. I hope you guys can help me out with some of my questions.

Do the melting temperatures of both the hybridising part of the primer and the entire primer with the extension have to be checked?. How close do the melting temperatures of the primers (hybridising+entire) have to be? I didnt quite understand why you have to check both temperatures.

I also wanted to know how far from the start codon of the gene can the upper primer be? I am having trouble designing stable primers because of the location restriction.

Also how low do you go with your Tm ( melting temperatures) for the primers (just the hybridising part)? One of mine goes till about 42 degree celsius.

And also what is the average length of a primer? For mine just the hybridising part are an average of 20 base pairs.

One last question, how do you design the 5' extension for the primers? Do you have to maintain a specific length for the extension?

I would be very grateful if you could help me out with this.

Im not an expert in all of this but I tell you what I would do it if I were you.

I will design my primers to anneal the DNA sequence with a Tm of 60 degrees. Then I will add to them the restriction sites I need leaving some bp in between as recommended in this website:

http://www.neb.com/nebecomm/tech_reference...ed_vector.asp#B

This is because the restricion enzyme activity can affect up to that point of base pairs and you don't want RE to modify your sequence of interest.

About taking into account both Tm, I will initially perform a PCR with 5C less than the Tm of your primer without the RE added bases. If you dont get unspecific bands, then it should be good enough. If you get some unspecific bands you can do it at that initial Tm for 10-15 cycles and then uses the entire Tm.

When desining primers I will pay more attention to the Tm I get than the lengh of your primers.

Look the website I mentioned above because they give you good advice in many of these aspects you are asking.

good luck