I am writing to you regarding a problem I had with my sequencing results. Allow me to describe the situation for you.
I cloned 79 pb of MPITS1 into plasmid TOPO10 and the colony-PCR results were positive. But the issue is that when I send it for sequencing, I got the results with double insert, that means for me that my sequence of interest was twice inserted into my vector which is pcr4-TOPO. I repeated it 4 times (The vector weight is 10ng/µl with 413 pb and my insert is 44ng but I used 1:5;1:10 then I diluted until I got 1ng/µl and I used 1µl of insert for 1µl of vector), unfortunately the results were the same. So, I decide to contact for looking into why my experimentation is not working. I was hoping someone could share some insight as to whether I should change something or not. Also, I was wondering whether I misread the results. I would be grateful if someone could find time to answer my question.
Looking forward to your reply,
LA
I think you should design primers and ligate based on RE sites...rather then using T/A based cloning method.
i would try diluting the insert further so that it is more likely to find plasmid to ligate to rather than other insert molecules and pcr across the insert as a quick method for determining the insert size eg plasmid dna +79bp or plasmiddna +158bp by direct pcr of a scrape of positive colony without purifying the dna just a few cells in the pcr mixture
May try to increase the screening number for transformants by colony PCR and one cut restriction digest to get the right transformant.
I recommend to increase the insert dilution as well as the vector/insert ratio. On the other hand you may screen larger number of colonies using the colony PCR.
Dear Codjo,
Design your primers having restriction sites at 5' ends of both the primers. Restriction sites should be selected on the basis of restriction sites in the MCS region of the vector and according to the orientation of the insert. Try ligation at 1:5 and 1:10 (vector to insert) ratio. Screen your transformants by Colony PCR.
Hope this will help you.
Feel free to contact.. if you face any difficulty...
Good Luck.
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