I transformed XL1B cells by cloning 16S rDNA gene of a thermophile strain to pTZ57R/T vector. The cloned plasmid vector isolated from white recombinant colonies were quite good in the gel, and the restriction digestion via Sma I also appears to be of the correct band size with the insert. But due to unavailability of other restriction enzymes available in MCS region of the plasmid vector, I wasn't able to isolate the inserted gene to produce it in the gel. Should it go directly for sequencing, or should it be further confirmed by digesting it with the other restriction enzyme?
Dear Sarbesh,
Restriction digestion is primary, It is always good to confirmed by sequencing, the pTZ vector has T7 pormotor site or other common site (check map), the sequencing company generally has this kind of primer. So just send your recombination vector to them.
you must sequence . The restriction enzyme checks only one specific site in the insert but you may have generated Taq polymerase induced errors in your insert elsewhere in the sequence and digestion will not identify these
I always do both, restriction digest and colony PCR to check for the insert being what I am expecting. Then I have to confirm with sequencing. So if you are not sure that you have your insert in the construct and you don't have the desired enzymes, why not go for PCR and then sequence!
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