pQE30 Vs pET - Which is the better? (Mar/03/2005 )
I am having problems cloning my expression genes in pQE30 vector.
So, I am planning to change to pET vector.
How easy or difficult is it to clone in these vectors?
I worked with both, IMHO they are quite standard vectors, cloning in neither gave me any unusual problems. should work for you, too!
I have been trying to clone 2 of my expression genes into pQE30 vector from past 6 months, and with no results.
At first, I had designed my primers with Bam HI site at both ends and when I cloned and transformed the ligation mix into E.coli, I got only one clone, but in wrong orientation.
So, I designd my Rv primer with SalI site. But now I am not able to get any positive clones.
I digest both my vector and insert, dephosphorylate the vector.Run 2ul of both vector and insert on the gel to know the concentration.
I have used V:I= 1:3, 1:1, 3:1 for ligation.
I will be making some fresh competent cells, since the earlier ones had very low transformation effiency.
Could someone plaese help!!!!!!!!!!!!!!!!!!!!!!
Facing problem with pQE30 Expression vector.I am not getting my protein. Do you feel change in pET28a expression vector will give me some edge ?
is it just your protein that's not expressed or is the expression system not working at all (i.e. have you included a positive control)? there are lot of possibitlites for a protein nor being expressed, ranging from codon bias issues over small proteins being rapidly degraded and wrong expressionsystems to just simple errors (like: did i put a startcodon in my seqeuence or not, hm, maybe i should check). so basically i can't recommed to you a specific expression system over another, i just can tell you, that IN MY CASE, the pQE30(31,32) and pQE40 worked very well...