I had cloned a gene into pET28 and transformation was done on E.Coli DH5a. After transformation, the colonies were grown on Kanamycin plates of concentration 50ug/ml. The colonies were then picked and inoculated in broth for isolation of plasmid. But when the isolated plasmids were run in 0.8% agarose, I did not get any bands. The isolation procedure was repeated thrice but in no vain. Why are the colonies and how?
Is there any colonies in control plates?
or try to take more no of colonies for plasmid isolation.
Sir, I had a positive and negative control that are fine. I had inoculated the colonies in 5 ml broth each that was totally used for plasmid isolation. So, everything is fine with that. @ Abhilek Nautiyal
Hi Santanu,
First, do a PCR (using overnight culture as template) with the primers for the cloned gene. Select the PCR positive clones to proceed with plasmid extraction. PCR results will tell you if the colonies that you have picked up contains your plasmid of interest.
I had added antibiotic of same concentration to the broth too. @ Abhilek Nautiyal
I had conducted colony pcr but the answers were negative. But my question is, if the plasmid of interest is not transformed why am I getting growth in my plates. @ Kiran Sreeja Jayan
Negative PCR results suggests that those clones don't contain plasmid+insert. however, it doesn't mean that they don't contain your plasmid (JUST THE VECTOR ALONE- NO INSERT!!!). In your case, you can not extract the plasmid > that means there is no plasmid or something wrong with your extraction kit! How about your control plate? Also, is your antibiotic working?
The plasmid isolation protocol is fine as it worked fine with positive control. The antibiotic is also fine as no colonies grew in negative control plates.
okay! What did you use as negative control? Digested plasmid and water?
I did not add anything to the negative control!!!! Its just a kanamycin LB agar plate. @ Kiran
Just LB+Km plate? that is not negative control for cloning! Just LB+Km plate doesn't tell you anything! You should have transformed digested plasmid + water (sterile) into the cells and spread the culture on LB+Km! If you get a lot of colonies on your control plate, which is the digested plasmid+water, cloning was unsuccessful (or the probability is very high). In your case, you don't have a control!!! So the colonies on your spread plate are either spontaneous mutants or cells with the plasmid alone (undigested plasmid - no insert, could be due to plasmid re-ligation or partial digestion).
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology