We often perform Quikchange mutagenesis on cDNA and send our samples to Genewiz for sequencing. Lately several of us in the same building consistently get "no priming" results for almost all our plasmids. We purify the DNA with the Qiagen miniprep kit, and calculate the DNA concentration using a nanodrop to confirm presence of DNA. Is anyone else having this problem? It may be an issue with either our Qiagen kit or genewiz, but I'm not sure.
We occasionally also get no priming results from Genewiz. Do you send you samples often on weekends? Generally, when I get bad reads its associated with samples being sent over weekends. I now try to avoid this. I know they send the samples to NJ for processing and I don't know if the DNA is kept under stable conditions for the duration of the shipping. We also tend to send a little more DNA than what they request, this might be something you try. We also use Qiagen and get good clean DNA by gels, so I don't believe it is a kit issue. Make sure that your DNA has the site you are attempting to prime for sequence, I am sure you have, but we don't normally see most of our plasmids return a "no priming" result.
Hope this helps and best of luck. Let me know if you have any further questions.
I am routinely working with sequencing task in my lab (AB system - Sanger method). If you sure about existence of plasmid DNA, you may consider about sequencing primer. Did you send primer or the company prepared it? DNA quality in 1 or 2 weeks sure not problem.
Hi Ali,
It sounds as though the samples are producing no results and Genewiz believes that no priming is the issue. If this were an isolated issue instead of something occurring with other researchers in your building then it simply could be a factor of no priming. If it is a problem with the Qiagen kit, I would suspect other researchers are using the same kit (or lot number) as you are if they are all having the same problem.
However, no priming may only be one possibility.
The DNA could be nicked during isolation. Nicked DNA generally will not sequence. The best way to detect nicked DNA is to load on an agarose gel. For plasmid preparations, you should see 2 bands. The faster moving band would be the good supercoiled DNA. The slower moving band is the nicked DNA. Most plasmid preparations usually produce some nicked DNA. If you see a single band, it is probably nicked.
Of course, as others have mentioned, the priming site could be missing. A single base change can effect the ability of a primer to sit down on the template.
The primer also could be bad unless you are using a universal primer supplied by Genewiz. Does this particular sample have universal priming sites that you can use to confirm the template?
There are other possible causes. But it is difficult to tell without trying to perform sequencing on the sample. If you would like, you could send a couple of samples to us and let us work with them. We also have a universal primer that sequences any plasmid with ampicillin resistance. We have a similar primer for kanamycin Maybe we can find the problem and there would not be any cost for these samples.
Let us know if we can help.
Doug
Hello,
We are experiencing the exact same problem in our lab. Researchers on completely different projects are suddenly getting bad sequencing results from Genewiz on DNA templates and primers that, in the past, have provided excellent sequencing results.
We are wondering how you resolved this issue. We have tried contacting genewiz however they have not provided any helpful answers. We are currently looking into sending sequencing samples to new companies.
It would be great if you had any advice on how to resolve this issue from your experience.
Aileen
Hello all,
I am getting back 'no primer' and 'poor quality' results from Genewiz, for several different batches of sequencing. Have any of you discovered an answer for why this occurs?
Thanks,
Rachel Burdorf
I've also had this problem, which is a first for me - my sequencing results used to be perfect in my previous institution where we did in house sequencing, now I'm lucky if 1/20 is decent when I ship my stuff off to GeneWiz. The main reason I use them in the first place was because they were recommended to me by a colleague. I recently got fed up of the 'no priming' issue and have decided to send them my gDNA samples and the primer data. Outsourcing both the assay development, PCR, clean up AND sequencing is going to cost me a fortune but if I get back poor quality data this time, I'm done with this company.
I have been using their UK service for Sanger Sequencing once with full success. Now, when relying on them for the second time, I got 'No priming' errors for 4 out of 4 samples, 8 out of 8 reactions.
However, both the first and the second set of samples do feature the very same backbone and I chose the same forward primer. The absorption ratios looked great, thus indicated negligible sample contamination with Gua-salts. I am using nuclease-free water stored frozen at -20°C and always use gloves and filtered tips, thus contamination is very unlikely.
I am looking forward to reading your experiences - and would be even more delighted to know how to deal with this!
- Badges
- Science method