hello
I am working on subcloning to insert my target protein to new vector, after the restriction, ligation,and transformation I did sequencing for the DNA by the vector primers VP1.5 nad XL39 and the result was fine the protein is inserted but there is stop codon between the protein coding sequence and the tag need to be removed. I used the site directed mutagenesis kit to remove the stop codon then the product was transformed and sequenced again
the XL39 primer sequence showed the stop codon was removed because it is close to the mutation site but the VP1.5 primer sequence failed (no reading at all with big blob) for all the 5 colonies that picked
the cycling parameters as the kit protocol suggests; 25 Cycles, Initial denaturation 98 °C 30 seconds, 98 °C 10 sec, annealing 63 °C 30 sec, 72 °C 30 seconds/kb -the vector size is 6200 b so I extend for 4 min- Final Extension 72 °C 2 minutes
Is there any explanation for this?
So, judging from your situation, my suggestion is to change another sequncing-primer on your plasmid. Is there any more applicable start point? whether in clockwise and anti-clockwise direction. In your experiment, it's impossible to check whether your mutagenesis successful or not by Gel. Sequencing seems to be the only method applicable. Whether the PCR readout is clean? I mean the band is obvious without any unspecific banding readouts? Best wishes on your experiment. Or can you just keep going on to check whether you can get the full-length protein? I mean, if the stop codon make sense, you cannot achieve the full-length protein but the truncated one, which cannot be pull-down by the affinity tags (e.g. 6-His and etc.). If you can get the protein with right molecule, you needn't to be trapped by this problem since there're really a lot of reason for sequencing failure.
Best wishes on you experiments.
Dear Ying
I did the sequencing with another primer and the sequence failed again
I think the problem with the cycling parameters, I will repeat the subcloning from the beginning
thank you
Hi Wedad,
It is not always necessary to follow the cycling parameters of the kit. You can try different parameters and can get a better result. Try changing the elongation time per kb and no of cycles.
Best of luck!
just getting a blob suggests that the primer is not annealing so either its sequence does not exist exactly or the annealing temperature is too high. As another primer has not worked you could just try a much lower annealing temperature ( the target size is small so there is probably nowhere else to anneal) or if you think that tertiary structural problems are to blame then sequence in the presence of 1M betaine and then very thoroughly desalt before loading on the capillary. If you posted the original .abi file there is often embedded information which might help
- Badges
- Science topic
- Similar topics
- Linguistics
- Composition Studies
- Publications