I am trying to clone 5'UTR of a gene in pGL3 basic vector. I have done PCR of the construct and getting positive result. Howerver, when I digest my clone with the enzymes that were used to create it , I am not getting any band for insert. I am using Kpn1 and Hindiii enzymes and 2.1 buffer of NEB
I assume you added the recognition sequence to the 5'-end of the gene specific primer for manipulation. How many extra bases did you add to the upstream of these primers? A few bases at the 5'-end of primers sometimes get lost during the PCR and if you did not have any extra bases, the recognition site may be lost with the deletion.
I am guessing you are doing colony PCR by stabbing colonies on your transformation plate. In this case, you probably have DNA contamination from the ligation mix that was also spread on the agar surface when you spread your cells.
Whether you did the PCR by using colonies as a template or plasmid.
I think better to use your plasmid as PCR template and check again if you could amplify your insert.
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