Primer designing for expression vector

[Emerson Martins]

I solved a similar problem cloning from a regiom upstream of the ORF, and
then with the plasmid purified making a nested PCR with full ORF. It
works...

2009/9/16 yoginee budhkar <eenigoy from gmail.com>

> Dear All,
>
> I am planning to clone a gene of interest in qiagen expression system,
> using
> their vector pQE30 UA, in which directly the PCR amplified product can be
> cloned, just like the good old TA cloning. The design of primer should be
> in
> such a way that the start codon should form the first 3 nucleotide of the
> insert DNA. There can be no manipulations there!
>
> I have thus thought of taking 1st 23 nucleotides The problem is with the GC
> content of this primer. GC is just about 35%. I can manipulate the reverse
> primer since it is N terminal His tagging vector, therefore the ATG has to
> be in frame with the vector's other cis elements.
>
> Any warnings that I should be considering in particular?
>
> --
> --
> Yoginee Budhkar
> Senior Research Fellow
> Food Engineering and Technology Department
> Institute of Chemical Technology
> Matunga, Mumbai 400019
> Contact: +91 9223355677
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