Problem with Transforming E. coli DH10Bac - (Mar/27/2012 )
I have been trying to transform E. coli DH10Bac cells with a pFastbacHTb vector containing my gene of interest, as a part of a protein expression experiment in Sf9 insect cells (BEVS; Invitrogen).
Since I don't have access to competent DH10Bac cells, I tried to make them heat-shock competent by CaCl2 and glycerol by a widely-used protocol, using DH10Bac cells from 2 different sources. However, in my last 3 attempts, I couldn't get any colony on LB-Km-Tet-Gent-Xgal-IPTG, 48 h post transformation which is supposedly the required time for a homologous recombination to happen inside the host (+ its helper vector).
I also used InsTAclone PCR Cloning Kit (Fermentas) which usually works well for transforming E. coli cells. However, using this method, still no clone at all appeared on the plates.
I was wondering whether I could use "electroporation" for this purpose. Does anybody have any experience with electroporating DH10Bac cells?
Any tips regarding the above-mentioned heat-shock or chemical transformation protocols or electroporation would be deeply appreciated.
I use the same protocol to make my comepent cells and it works absoloutely fine. DH10B cells are also very stubborn cells, so I don't think you really need to electoporate them. Wait for other users to reply, I think the user 'pDNA' must be more helpful than I am, but I'm guessing you must be doing something wrong during incubation with CaCl2 and addition of glycerol. I also have this terrible experience that whenver our -80 freezer breaks down, my cells don't give any colonies anymore. They are very temeprature sensitive.
Do you have a prepared plasmid, or is your transformation from a ligation reaction? The competence of CaCl2 prepared cells is quite low, and not suitable for transformation after ligation. Also, do I understand that your plasmid has resistance to Tet, Kan, and Gent? Isn't that a bit much?
Thank you Curtis and phage 434 for your responses.
Phage434: I have a prepared plasmid (miniprep) and not a ligation reaction product. The plasmid (pFastbacHTb+gene of interest) is resistant to gentamicin. The host itself (DH10Bac) contains a bacmid and a helper plasmid which are resistant to kanamycin and tetracycline, respectively. After a successful transformation, the final bacmid product should be resistant to above 3 antibiotics, as a result of homologous recombination.
So, the transformation must not only get into the cell, but also undergo recombination to make this work? This sounds like an inherently low probability event, and probably demands high competence. I'd go for electroporation.
Yes indeed. But has anybody out there actually done electroporation with DH10Bac cells?
As far as I know, most protocols recommend the heat-shocking method and I've encountered a few notes which advise against electroporation for this purpose.
Theoretically, I don't see why electroporation may not be suitable. I am asking these (instead of just doing it!) because I need to make the cells competent and borrow equipment in advance while I am crunched for time and I may instead optimize the heat-shocking steps.
Well, if electroporation is difficult, you could make your cells competent. I'd recommend either the TSS approach, or the CCMB80 approach, both of which will give cells dramatically more competent than CaCl2 approaches.
Thank you for the info.
hi everyone, i have been facing the same problem as IPI. I can't transform the recombinant pFastBac HT-C into DH10BAC cells while my positive transformation control, pUC19 can be transformed into the DH10Bac cells. My recombinant plasmid (insert+pFAstBac) is 5996 bp in length, while pUC19 is ~2.6kbp, is it possible that the recombinant plasmid is too large to enter the DH10BAc cells? If this is the case, what would u all suggest?
And IPI, did you solved your problem? What factor did you find that could improve this problem?
Thank you everyone!