DNA sequencing troubles

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Knowledgehound
Knowledgehound's picture
DNA sequencing troubles

I am having trouble with my sequences in that I get NO results from sequencing. The control that comes with the sequencing kit (BigDye v3.1) on the other hand is always present. This tells me that I am not loosing my templates in the precipitation step (I use ethanol and sodium acetate for this). I also run gels for my two PCRs, so I am sure I have template when I go to the cycle sequencing. I have tried using primer designed for sequencing and also primers used in the previous PCR steps even at different concentrations and still, NOTHING!!  The cycle sequncing conditions are 96deg/10s, 50deg/5s and 60deg/4min for 25 cycles. I have troubleshot in about every which way! Could anyone point out what I might be missing because I am at my wits end...
Thank you.

Ivan Delgado
Ivan Delgado's picture
Hi Knowledgehound,

Hi Knowledgehound,

From your description it sounds like you are able to get DNA sequence to work when using the control that comes with the kit. This of course suggests that everything is working, from the PCR to the DNA sequencing. Because you've run the necessary controls, it is possible that the problem is your PCR of interest. You may be getting a PCR product, but the PCR product may not be what you are interested in. In other words, you may be getting amplification, but the amplified product is something else. This would explain why the DNA sequencing reaction does not work (i.e. your primers "barely" amplify a PCR product, but when you use the same primer to perform the DNA sequencing reaction they do not work). My suggestion is to try a different set of primers for your PCR and run your DNA sequencing reaction again using these new primers on this new PCR product. As a side I assume you tried to run the DNA sequencing reaction using both PCR primers (5' and 3') and neither gave DNA sequencing results. 

Hope this helps.

Knowledgehound
Knowledgehound's picture
Thanks for your feed back

Thanks for your feed back Ivan.
Yes I did run both 5' and 3' primers and none gave a sequencing result.
I'll try a different set of primers like you suggested and see how it goes.
Thanks.