hi everyone,
i am trying to figure out why the gene inserted is not amplifying after cloning. I have inserted a mycobacterium-specific gene into pET 32a plasmid and transformed the ligation product with BL21 cells. after transformation, I have isolated the plasmid from the obtained clones and done restriction digestion with the desired restriction enzyme. i have got a positive result for this experiment by running the restriction digestion product in 1% agarose gel. hence i kind of confirmed that the cloning has worked. ionrder to re confirm it, i have done a colony PCR with the obtained transformant colony.but i have got no amplicon for the gene. i have also tried to amplify the gene from the isolated plasmid using the gene-specific primers,but that also gave a negative result. i have repeated these experiments for multiple time but each time am getting the same pattern of result. that is a positive result for restriction digestion of the isolated plasmid and negative result for the colony PCR as well as the plasmid PCR. what can be the possible reason behind this. also i tried to express the protein using iptg induction, that also resulted in negative result.
The PCR did not work (on the plasmid and on the colony), the expression also did not give any protein.
Potentially, the DNA fragment you cloned is not your target gene, but another DNA fragment you cloned.
If you want to confirm your result 100%, you would need to carry out sequencing.
Hi,
I think that there could be one of the problems below:
1, your PCR conditions. You should make sure your PCR conditions were optimized.
2, PCR primers
3, Contamination of the PCR reaction mixture or DNA template
4, The vector after cloning contains mutations
5, Size of the gene
I hope this information is useful for you.
Good luck!
Sofiane Benyamina thank you for the valuable suggestion. but the chance for cloning another DNA fragment is very low because the gene as well as the plasmid we used were purified from the agarose gel after restriction digestion and subjected to ligation. also, the fragment we have obtained from the restriction digest of the cloned plasmid has an approximately similar size to the desired gene. As you suggested, we have sent the plasmid for sequencing.
Dear Swathy S Anand
just some questions:
how long is the insert? Which primer did you use for amplification after cloning the insert specific primers used for the PCR or the T7 promoter and T7 reverse present of the pet32 backborne? Which polimerases did you used for colony PCR?
best
Manuele
Phan Thi Lam Hong thank you for the response!
To rule out the chances of contamination we performed another PCR reaction with the genomic DNA as the template as a positive control and that worked perfectly. So the contamination in the PCR components and the conditions may not be the reason. Also, the size of the gene is only 670bp.
Manuele Martinelli the insert length is 670bp and used the gene-specific primers and and GeNei red dye PCR mix which contains Taq DNA polymerase.
I also vote for sequencing your insert. Colony PCR can be a bit finicky and it's important to ensure your insert is correct and doesn't have any errors that would interfere with proper protein production.
Colony PCR is notorious for having false positives (and false negatives). My advice for cloning is to number & streak out several colonies on new selective media. Set up an overnight liquid culture with selection, then purify your plasmid. You can even make a freezer stock of the liquid culture just in case it's needed later.
And sometimes you can't use an intact plasmid directly for PCR. Usually due to secondary structure in the molecule. Find a restriction enzyme that will linearize your plasmid (make sure it won't cut in your insert!). Linearize & then do PCR. Or send the plasmid for direct Sanger sequencing.
Good luck!
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