can there be results where a colony PCR showed a positive result to a gene cloned in a vector ; but after restriction digestion of that same recombinant clone; there is no band of that particular gene size - why
It depends on the primers you used to determine if the target is present.
If they are internal to the gene then when you plate the bacteria and also the plasmid on agar if you take a negative colony for pcr then the wetting effect of the reagent mix will give you an amplimer so making the negative clone look positive. When you grow on the clone then the wetting effect is diluted so the pcr or digest will also be negative. For colony testing the best primer set is one primer on the plasmid and the other internal to your gene then only transformed colonies will give a pcr product
Hello,
To check make a PCR on colony of an unprocessed bacteria with a recombant DNA and look if you do not get an amplification of a fragment that is the size of your gene, if this is the case that it is that it is A parasitic amplification.
Also you can recover the recombinant DNA and without enzymatic digestion, check directly its size by electrophoresis and look if its size corresponds to the size of the vector + insert.
Yes, there is a possibility of positive results in colony PCR to a gene you cloned into a vector, but no band in restriction digested product at the expected size of your gene. For this problem, you have to follow a few troubleshooting steps
1. Check your primers and other reagents you use for PCR if they are giving bands at your gene size without adding any template.
And if the PCR didn't give any band, go for the next step.
In case you get a band in no template PCR reaction change the reagent that may have got contaminated with your gene while operating.
2. Check whether the restriction enzymes you use for the experiment do not have a cutting site in the gene you expect. If the gene has any cutting site then your problem will be solved by selecting other restriction enzymes which do not cut your gene.
It is better to check for the number of cutting sites and position of the cut for your choice of restriction enzymes inorder to calculate the expected band size after digestion. Deepshikha Keot
thank you all for your valuable suggestions
@Jammugani Vinod Kumar
@Sofiane Benyamina
@Paul Rutland
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