PCR Amplification of Insert and Restriction Sites Problem

[admin]

Hi, I have a question from an email:

Fortunately i got the cDNA of the gene cht2 that i work on after along time, but now the problem is i want to clone in to pichia pasatoris so i have to insert RE site in my gene.

That site is Cla1 & Xba1 ,but every time i amplify the gene using primers
with these RE sites, i couldn't get the RE site .i change the primers several times and the same result each time.i hop if anybody can suggest something useful to do it?

thanks!

[knockout maker]

Hi there!
In general, clavage of PCR products is inefficient due to the inability of some restrictions to cut close to DNA ends;it is therefore best to add several bases to your primer to act as a stuffer. I generally stick 8 to 10 bp Guanine tails but, depending on the enzymes, sometimes even more(this is particularly true for some enzymes like NotI or XhoI). Alternatively you could just subclone your fragment into pGEMTeasy or TOPO or another comertially available vector for easy subcloning and then cut you insert with ClaI and XbaI. Many laboratories do that routinely and never dare to even try to cut PCR products directly for subsequent subloning. You can find how many bases need to add for the stuffer depending on the restriction enzymes you are using at the new england biolabs website. Cheers!!

[admin]

Thank you that was very helpful knockout maker

I also found a reference on difficulty in digestion of restriction enzymes from the end of a DNA molecule. It helps as you mention to have several nucleotides every at each end (4-5+).

Cheers!