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?
Senior Research Fellow
Food Engineering and Technology Department
Institute of Chemical Technology
Matunga, Mumbai 400019
Contact: +91 9223355677