Dear researchers,
I have coloned my gene of intetest in pET29 and confirmed it by digestion and pcr from the miniprep. Interestingly, sequencing failed when i use my primers. However, it reads when i use T7 promoter primers of squencing company. The disappointing fact is that the resulted sequence only matches expression vectors on the database. should i assume that for sure my nsert is not there?
Thanks,
Mah
did you get more colonies in your vector+insert+ligase than in the control sample vector+ligase sample?
I am wondering if you have cut the vector and the positives are vector incorporating into vector because for some reason the insert dna has not inserted
Thank Paul,
There was not that big difference between the plate which contained insert and the other plate but yes in total i had more colonies.
PCR and digestion have confirmed my insert is present and i did it more than once.
I believe that the glycerol stock which i prepared from the original bacteri which showed positive result has lost the insert for some strange reason. Because pcr doesnt show my insert there either!
Do you have any idea how and why? It was only one week old stock.
Thanks,
I cannot think of a good mechanism for this but somehow you may have created a resistant strain which does not have your insert and you may have frozen a mixed population,I would grow up some of the glycerol stock, pcr from vector to insert and if it is positive plate out the mix again and colony pcr to get the insert strain back,
good luck
Paul
Thanks again Paul. Yes, i am gonna do what you suggested. I just got the sequencing result of my PCR product and it is what i look for. So now i should make sure about my stock.
Best,
Mah
an afterthought Mahbobeh...what if your cell line was transfected with insert vector and non insert vector at the same time on transformation. then it would be antibiotic resistant but still have native vector which sequences short?
Paul
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