Posted 27 September 2011 - 03:11 AM
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?
Posted 27 September 2011 - 06:21 AM
The bigger question is, were all the colonies you picked correct or did you also have some vector background?
Best of Luck.
Posted 27 September 2011 - 12:48 PM
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