I am performing ligation of the plasmid and a target gene. The steps I have taken are:
1. Double digestion of the plasmid and target gene
2. Ligation of the plasmid with the target gene
3. Transformation into E. coli
4. Screening on LB+ampicillin medium
5. PCR with specific primers
The screening results on LB+ampicillin showed good results. I also performed plasmid isolation from the growing colonies. The plasmid isolation results were electrophoresed and showed bands corresponding to my target recombinant plasmid.
However, I encountered a problem during the PCR step. I performed PCR using primers specific for the target gene and primers specific for the plasmid. The PCR results are shown as figure follows bellow.
The first three wells displaying bright bands are the PCR results of recombinant E. coli carrying the plasmid without ligation, while the remaining wells are the PCR results of recombinant E. coli carrying the plasmid ligated with the target gene.
I wonder why the PCR results do not indicate that the bacteria carry the recombinant plasmid I inserted, while the plasmid isolation results show a band that matches the size of my target plasmid. Any suggestion on which step I should be further evaluated. Thank you
It could be two things: A. your plasmids are all empty parent plasmids. run the parent plasmid( without digestion) and your extracted plasmid Side by side, if there's insertion you should see a size difference; B. your primers are not working. run a positive control PCR with the target gene as templet
Hi. I'm nafiseh paysokhan and about your question i think you should check out your electrophoresis buffer. if i understand, your problem is you have problem with clarity of your pcr. in our lab with had same problem, we did a lot of pcr and electrophoresis but under the gel doc we didn't see anything, after checking everything we understand the problem is the buffer and the ph of the buffer. so if you have a same problem i recommend you check your buffer.
good luck
Sometimes the secondary structure of plasmids makes it hard to use them directly for PCR.
Find a cheap enzyme that will cut the plasmid backbone, but NOT your insert (don't just guess or say "probably won't cut". Double check using the actual sequence).
Digest the plasmid.
Then use for PCR.
Good luck!
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