I often order genes synthesized and subcloned into pET28a at Genscript. Last time around I also ordered one subcloned into pET26b in order to add a pelB peptide sequence. Unfortunately, when I got my plasmids and transformed them into E. Coli I only got colonies from transforming with the pET28 plasmids. There were zero colonies on the pET26 plate (kanamycin plate). I did it a second time with the same results - colonies on other plates but not pET26 transformation. Why would the plasmid make a difference this way? What can I do to troubleshoot? Thanks in advance!
Hi Jonathan,
quick question : was your lack of colonies in DH5alpha-like cells or was it in BL21(DE3)-like cells?
I personally always put my new plasmids in DH5alpha to miniprep them, so that I have some long term archive of the DNA.
if you don't have colonies in DH5alpha, it is probable that there is a problem in the plasmid itself... a quick restriction analysis and gel, and a sequencing, could help investigate this.
if you don't have colonies in (DE3) cells, but do in DH5alpha, it might be that there is some leaky expression that turns out to be toxic for the cell, in which case there is a bunch of tricks that can be tried. adding a couple g/l of glucose to the media (recovery growth and plates) helps pushing down leaky expression a bit, switching to pLysS or even pLysE could also help. that putative leaky/toxicity could be caused by a set of bad luck leading to some secondary structure in the mRNA and/or some weird hybridisation. if you add the fact that you have the Rop protein in the pET26, maybe that has some compounding effects with metabolism...
When using strains other than NovaBlue : shake at 37°C (250 rpm) for 60 min prior to plating on selective media. Outgrowth is always recommended when using kanamycin selection. Shake at 200–250 rpm at 37°C for 1 h in SOC medium then put on kanamycin plates.
You can use a plasmid control for check your protocol (10 ng pUC19) and and maybe if no result a new batch of BL21 DE3 cell.
Hi Nicolas,
Thanks for your interest. Yes, you pretty much described how I transform the cells. I used outgrowth for 1 hr in LB media before putting on kanamycin plates. I am certain that it works because I get 30-40 colonies when using pET28a plasmids this way (these are my controls). There is a difference in the one that is cloned into pET26.
Maybe I should try miniprepping this plasmid in case it isn't pure?
Yes I think so too. It's a good idea, try to mini-prepare this plasmid and to deposit it on agarose gel and maybe in sequencing.
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