Designing cloning primes (restriction site and kozak sequence) (View forum version)



Richard.21

Posted 03 April 2014 - 07:18 AM

Hello everyone,

 

I am new in this cloning world and I need to design primers for cloning. In general I know that the forward primer must seem something like following:

 

FLANKING-Restriction site-Kozak-Start Codon-gene of interest-Stop Codon-FLANKING

 

Now I am wondering how should I design my reverse primer?

 

I would really appreciate any help and tips to primers designing for cloning

 

 

Regards


bob1

Posted 03 April 2014 - 12:56 PM

You have it a little wrong there... the forward primer would not typically include the stop codon, that would be in the reverse.  Restriction sites etc are always added to the 5' end of the primer.

 

So normally you would have 5' 6bp (for restriction annealing)-Restriction site - kozak - the first 20ish bp of the gene of interest (including ATG). 3'

 

The reverse would be the

flanking (6bp)-restriction site - reverse complement of the last 20ish bp of the goi (including stop codon)