My PCR product always lost the last 10bp and also the digestion site, which made my ligation never work, can someone help me with this problem?Thank you so much!
You need to post more details. For instance, how do you know that the PCR product lacks the restriction site?
Do you see this error when you sequence the PCR product itself, or are you cloning into a plasmid, sequencing and then finding the error?
Yeah, I am really sure, because the ligation never work for both T4 ligase and infusion cloning. so I sent the PCR product for sequencing, then I found that the reason was that the homologous part and the digestion site was lost, even the last base pairs of my gene, so that's really strange.
Does not really make sense. Amplification is happening so primers are working. What is the length of your primers? Number of nucleotides that hybridize to the template and number of nucleotides that hybridize to vector?
You will not see reliable sequencing data for the primers or within a number of bases from the primers. If you are using the same primers for sequencing as you are for introducing the restriction sites, you will never see them in sequencing. Your cloning problems are due to something else.
My total gene is 1600bp, in order to check whether sth is wrong at the end of the fragments. I designed two primers ,the first one is at about 300bp of my gene to get the 5' part, the second primer is from 1400bp try to get the 3'end. then I sent the PCR product for sequencing with these two primers, after check, the 5' end is totally fine, I can see the restriction digestion site, the overhang. But for the 3' end, the last 7 base pairs and the restriction digestion site, the overhang totally lost. so that's the problem. I really donnot know what happened.
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The primers you are using to amplify the gene must be long, how long? What is their purification grade?
Ah, that's very helpful.
Only thing I can think of is that the original 3' end primer was bad. Physically, the 3' primer would have to be present in all PCR products made from it. Have you looked at the raw sequencing peaks to see if it looks like there are competing sequences in that region, or does the data just stop after that? If the former, the template could have a population with a frameshift at the 3' end. If the latter, may also want to check the 3' primer for self-annealing if you haven't already.
check the paperwork that came with the oligo from the manufacturing company to make sure that the oligo is what it should be...sometimes a space or punctuation mark in the submitted sequence can look like the end of the sequence so a shorter sequence may have been made
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