Hello,
I have inserted a 580 bp sequence in 3xFLAG plasmid. This sequence has a 66% GC content. PCR annealing temperature was high at 68C. Purification, digestion were fine. Transformation was done in DH5a cells and were competent as well. I got a healthy number of clones and picked four of them. Clones were shaken overnight at 37C in LB media. Performed a PCR to check if the sequence was present. I got intense bands in all 4 samples. They were further processed by Axygen plasmid miniprep. I sent them for sequencing. I mentioned specifically that the content was GC rich. However the results indicated that "600bp of secondary structures" was found in 3 out of the 4. Now I am at a loss about what Im supposed to do next? Is it in any way possible to move on to transfection in mammalian cell line after such a result.
I have performed the experiment twice from start to finish, and both times my results mention the presence of secondary structures.
It seems to me that you can amplify and insert ok. If you were doing your own sequencing then you would use betaine or dmso to remove the secondary structure and it would sequence ok. You could find a company or department that you do the sequencing reaction and they run the sample and then you should get the complete sequence. Are you concerned that the secondary structure would mean that the transform would be inactive? The gene works in the natural state so it should be worth moving to transfection and see if it works on the basis that all sequences have secondary structure but I vivo they seem to work OK
It seems to me that you can amplify and insert ok. If you were doing your own sequencing then you would use betaine or dmso to remove the secondary structure and it would sequence ok. You could find a company or department that you do the sequencing reaction and they run the sample and then you should get the complete sequence. Are you concerned that the secondary structure would mean that the transform would be inactive? The gene works in the natural state so it should be worth moving to transfection and see if it works on the basis that all sequences have secondary structure but I vivo they seem to work OK
Thank you so much for your reply Dr. Rutland. Yes I was worried about how it would work when transfected. Your answer gives me relief.
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