Hi, I'm trying to transform E. coli DH10Bac cells with a pFB-Lic-Bse vector containing my gene of interest. 48 h after transformation, I get some white colony on LB-Km-Tet-Gent-Xgal-IPTG, but when I do the PCR using the M13 for and rev primers I obtain only the band relative to the bacmid alone. My recombinant plasmid (insert+pFB-Lic-Bse) is more than 10,000 bp in length. Is it possible that the recombinant plasmid is too large to enter the DH10BAc cells? If this is the case, what would you suggest? Do I have to prolong the transformation or use more DNA?
Thank you everyone!
It is normally possible to transform your cells with a plasmid which is more than 10kb. Are you using a ligation product or an already prepared plasmid? In case of ligation, most of the time, it is because of the ligation and not because of the bacteria. In that case, do you have controls without your insert with or without ligase?
It will be certainly a stupid question, but are you sure your cells are competent?
Are you using the good concentration of antibiotic? A too high concentration can prevent bacteria to grow, even if they have integrated your plasmid.
I do not have personally experience with DH10Bac cells. But the questions I asked above are often the reasons of failure. Otherwise, you can consult troubleshooting part of the manual. It can help sometimes.
Hi Valentina,
You are right in mentioning that as the plasmid size increases the efficiency of transformation will go down....What protocol you are using for transformation....In my opinion instead of increasing the time of transformation you go for alternative techniques which offer higher efficiencies of transformation such as electroporation...
bye
Thank you guys!! Of course I have already tested all the possible positive controls and checked the troubleshooting in the manual. Unfortunately, I can't try electroporation in my lab.
Thanks anyway..
Hi Valentina,
If electroporation is not feasible in your lab...go for some high efficiency chemical transformation methods (refer to any mol. biol lab manual e.g. Sambrook and Russel, 2003) instead of just Calcium chloride based method
bye
Hi,
I agree with all above nice suggestions.
Try heat shock method using water bath at 42 oC for 1min. follow proper kit protocol but you can increase time to 1 min.
you can try your gene specific primers as well as vector specific primers to check the proper clone and use different competent cells (having high efficiency of transformation)
Keep trying some modifications.
Regards,
- Badges
- Science topic