Lately I'm getting failed DNA sequences and strange sequence peaks in my chromatograms when trying to sequence my plasmid (see attachments).
With a previous mini prep sample, the following protocol did work (with the same primers). Now with the midi prep from the same sample/ and after a mini prep of an insert ligation into the same plasmid I'm having problems. I've tried this three times now. Very rarely I get short reactions (which correctly align with plasmid sequence), but mostly it fails. This is the protocol:
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For DNA I use purified plasmid: ± 500ng/ul; 1.90 260/280; 2.24 260/230. I also ran it on agarose gel (undigested and digested) and band sizes were as expected.
Primers: ± 20bp, Tm: 58-60C, 40-60% GC, I've avoided primers that make dimers/hairpins and I've checked if the primers have a single binding site on the plasmid
For the PCR reaction: 500ng DNA, 3.2 uM primer, 5x Seq. Buffer, 1ul BigDye 3.1, filled up with water until 20ul
Run PCR: 96C for 1', [96C for 10", 50C for 5" and 60C for 4']x25, 15C forever
DNA purification: adding to the PCR reaction 2ul EDTA 125mM, 2ul NaOAc 3M, 70ul 100% EtOH. Invert and incubate 15' @ RT. Spin down 14000rpm @ 4C for 20'. Discard sup and rinse pellet with 200ul 70% EtOH. Spin down 14000rpm @ 4C for 5'. Discard sup and air-dry for 5'. Resuspend pellet with 20ul HiDi form amide and incubate 2' @ 95C. Transfer on ice for 3' and transfer to 96well. Input in ABI 3100-Avant sequencer and run the machine.
Its not the machine since my colleagues do get good sequences and as far as I know we're using the same protocol.
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I'm now sending these samples to a sequencing facility to run the reactions. But any ideas on why my reactions fail?
I found the same problem once, it was contamination with another plasmid (with no insert). So, maybe transform again and choose just one clone to repeat the experiment.
Double peaks or multiple peaks are, generaly, caused by sequencing multiple products.
In your case, it is more likely that you have a problem with your primers. I would try a new stock of primers. If the problem persists, then I would look at the plasmid purification steps. If nothing works, start over from bacteria transformation (in some cases, bacteria can "edit" sequences inside plasmids, creating indels or even expeling plasmids containig repetitive sequences or AT-rich sequences).
Hi,
I agree with some of the answers above, however I have more suspicion on residual reagents after purification....I'll advice you to handle a control reaction yourself using same reagents and see if the quality is of same level...in your plasmid and control plasmid
Thank you all for your answers. I did perform another plasmid purification (midi prep), which showed good 260/280 and 260/230 ratios, but it didn't give me better results. After performing the same sequencing (with the same primers) on another machine (ABI 3730xl), it showed that the peak intensity dramatically dropped off after it had read a stretch of basepairs. It was always at the same point in the template sequence.
So, I resolved the issue in another manner:
After looking closely to the sequence I am interested in, I found it very GC-rich. What helped was alkaline denaturation of the plasmid + running the PCR with the dGTP BigDye Terminator v3.0 Kit. It was not successful with each primer, but about half of the primers showed good result which was sufficient for me to check the sequence.
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