I am trying to clone HA gene of influenza virus into the phw2000 vector. Top-10 cells were used for transformation. A few colonies tested positive using gene specific primers were processed for Miniprep and plasmids were sequenced. Sequencing results showed no insert gene inside.
After then plasmid DNA (after miniprep) was used and performed PCR using gene-specific primers which showed good band and PCR product is sequenceable which matched with the original sequence.
After then the sample was further processed for Maxiprep and sequenced. Results showed no insert gene inside because no peaks against the gene-specific primers.
Could anyone help me with this what I am missing?
Thanks in advance for the respondents.
It is often better to check for an insert using one gene specific primer and one plasmid specific primer. Then a positive result means that there is an inserted sequence. Gene specific only primers will give a positive result if either
1 there is insert pcr template present inthe cloning mix after cloning and that can amplify even if cloning has failed or
2 you have gene specific pcr contamination and what you are seeing is contamination. Are your negative pcr controls still not amplifying?
I am thinking that one possibility is that cloning has failed ( hence the negative sequencing) and the positive pcr is spurious and not created from the plasmid
It is often better to check for an insert using one gene specific primer and one plasmid specific primer. Then a positive result means that there is an inserted sequence. Gene specific only primers will give a positive result if either
1 there is insert pcr template present inthe cloning mix after cloning and that can amplify even if cloning has failed or
2 you have gene specific pcr contamination and what you are seeing is contamination. Are your negative pcr controls still not amplifying?
I am thinking that one possibility is that cloning has failed ( hence the negative sequencing) and the positive pcr is spurious and not created from the plasmid
I agree with Paul Rutlan, I used to that and If you really got good DNA band from PCR, you can simply cut that band from its electrophoresis gel and send for sequencing to find out what is it for. best wish
Yes. I agree with Paul. Those PCR bands might be contamination.
Have you tried PCR with one primer further within insert ?
Thanks all for your time and response. I agree with Paul Rutland. Although cloning has failed but still has the gene PCR template inside therefore giving a false-positive result.
To figure out what it is, I performed PCR of the same miniprep DNA/plasmid and used one forward primer of the gene middle location and reverse primer from the vector side and got band again which was sequenced. Results showed good peaks with gene forward primer and no peak with vector reverse primer, which is really strange to me. Mohammed Radhi Mohaisen Laurence Stuart Dawkins-Hall
It's really easy to get a false positive from colony PCR. Any "leftover" insert from your ligation reaction can be enough to amplify even though it's not transformed into your cells.
I'd test for positive colonies the "old-school" way: make a streak plate of potential colonies, grow up a liquid culture, mini prep the plasmids, set up a single-cut restriction digest, do PCR from the cut plasmid, send the PCR products for sequencing.
As my advisor told me "you can't rush quality".
Good luck!
Thanks Katie A Burnette .
Could anyone help me with the size of the empty vector phw2000, I found its size as 2980bp in one paper. When I run empty vector phw2000 on gel, it shows a size around 2kb. Does It show the supercoiling of the vector? Should I proceed with this vector? Please take a look at the gel photo. In labeling vector-N and Vector-O represents two different vector stock vials.
Many thanks for your valuable suggestions!
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