white colonies - (Sep/27/2011 )

Hi there,

I recently inserted my gene (the whole gene inc. signal peptide) into pbluscript and then plated onto amp plates for blue white selection...single colony pcred white colonies to see if insert was present and carried out with miniprep etc...

The next step of my exp was to clone only the mature form (so no signal peptide/Ribosomal binding site-to make a recombinant protein) and ligate into pbluscript and once again I did a single colony pcr...my question is how come this time no blue colonies formed?....is it due the the lack of RBS hence only white colonies will be formed regardless if IPTG/X-gal is plated?

Cheers,
b_06er

pBluescript should have the LacZalpha RBS and start site already in it, so unless you took it out of the vector portion, you should still have the possibility of getting blue colonies. It is possible that your cloning efficiency was so high that you only have positive clones, but I have only had that happen once. It is also possible that you used a different set of plates that didn't get either X-gal or IPTG or both, meaning all colonies would be white.

The bigger question is, were all the colonies you picked correct or did you also have some vector background?

Best of Luck.

Hi,

It most likely is due to not having X-gal/IPTG...The first time I created my plates with both of them the second time I used someone elses (that had amp but not sure if they already added xgal/IPTG--but I'll ask)...and yup, I picked a few and had quite a few positives