Oligo design strategy - cloning in pQE 30 (QIAGEN°) (Apr/22/2004 )

Hi all,
I hope that my questions don't bothering too much.

I would like to know "all your standard protocols" for cloning into an expression vector (like pQE), that introduce a polyHIS tag on the N-terminus.

I can figure that it should be very important using an appropriate oligos' pair in order to syntetise my interest fragment with appropriate features.

These are the elementary features that should be present:
a. Forward primer (20 or more mer): 15 bp completly annealing on template sequence (plasmid or cDNA); restriction site for the first available one in the multiple cloning site (in that case, I can choose for Sph I, so I have to add at 5' these bases: gcatg - I have already cheched that Sph I doesn't cut in elsewhere in the insert - Please correct me if I am wrong!).

b. Reverse primer (20 or more mer): 15 bp completly annealing on template sequence (plasmid or cDNA); restriction site for the last avaibale one in the MCS I choose for Hind III, so I add at 3' "agctt". Then I would like to introduce a codon stop (TAG for istance), in order to reduce the number of not desidered aminoacids.

I will appreciate too much if anyone of you would send me any suggestion.
Please don't hesitate to contact me for any question about this topic.

Thanks a lot in advance.

Kind regards,
davide

Hi KJ,

Most important thing for introducing restriction site into PCR product is that providing enough extra bases outside the enzyme recognition site in your primers. Different enzyme may require different number of extra bases to cut efficiently. Usually, 6 bases pairs on either side of their recognition site is needed for a enzyme to cleave efficiently. For a complete list of enzymes and extra bases needed, please see NEB site here http://www.neb.com/nebecomm/tech_reference...ized_vector.asp.
(Now the link works)

Hi KJ,

thanks for your prompty answer. I have already heard about the necessity of adding extra bases and I found that your suggestion should be very useful.

Anyway I think there is some wrong for the link you posted. Simply, it doesn't work; can you post it again?

I would like to know if I have to add those extra bases at 5' and 3' Terminus of the forward and reverse primers, respectively.

An other question: I need include into the primer design the entire restriction site sequence (for example: for Sph I site I'd add "gcatg", without the final "c" , 'cause these are the protuding bases, generated by this enzyme.
Am I correct?

Thanks in advance,

davide

Hi

you must add the complete recognition sequence of the restriction enzyme to your primer PLUS and additional number of bases at the 5' end of each primer according to this chart:

http://www.neb.com/nebecomm/tech_reference/
restriction_enzymes/cleavage_linearized_vector.asp

(had to divide the link, wouldn't work in a single line...)

So for your Hind III SphI digestion you should add 2 bases at each end, which should give you more than 90% cutting efficiancy for both enzymes.

And of course you can add a stop codon to your reverse primer, shouldn't really matter. But I think the pQEs have stop codons in all three reading frames just after the MCS, they should do the job as well if you don't mind the two or three extra aminoacids at the c-term of your protein...

Mike

Hi...

Also, be aware to keep the open reading frame of your DNA target after you add some bases. The addition should make no change of ORF of your DNA (PCR product).

Good Luck