I stored my amplicons in -27 celcius and they are already purified to be used for Sanger Sequencing. However, my sequencing results were very poor, the sequence were almost not detected at all. I've tried twice with different volume of samples but the results were still the same. I wonder if my amplicons had bad quality.
Before you send for sequencing you need to be sure whether your PCR amplifies you region of interest with sharp bands on agarose gel, which also should be RNA free preferably.
You can troubleshoot from the scratch:
1. Check the DNA quality.
2. Reamplify using gradient PCR is annealing is not correct.
3. Make sure you get dark bands at 30-35 cycles
If all things are well you don't need to worry about storage, 4 degree will also work. I had sent my tubes in box with few ice cubes.
One more thing you can do after you are thorough with all steps is, prepare the master mix of more volume then needed for sequencing. Run extra sample on agarose to confiem amplicon quality.
Hi Juli,
For good quality sequences you need:
1. good quality amplicons - I suggest you run at least 4 μl of your purified PCR products on an agarose gel ; if you can also add a low DNA mass ladder in one lane would be even better because you will also have a quick estimate of both quality and quantity of your amplicons. If you send these out for sequencing you can also include a picture of your gel so the tech preparing your reactions can guesstimate how much template to use.
2. good sequencing primers - some primers may work fine for PCR but not as well when it comes to the sequencing reactions. Make sure you have there correct sequencing primers.
3. good storage conditions - although amplicons can be kept at 4ºC for a short period of time, sequencing reactions will always work better with fresh amplicons or amplicons kept at -20ºC. Thawing and freezing these amplicons too many times may also induce degradation.
This should cover the troubleshooting as far as sequencing templates are concerned. If you are also setting up your own sequencing reactions let me know and I can walk you through that protocol.
Good luck!
Simona
First of all you need to check integrity of your amplicon by electrophoresis before sequencing. We need to know if it is the sequencing process, not DNA quality, that interfere with your results.
I agree with Simona. Check the purified products first. As a matter of chance, your PCR clean up kit buffers (especially the elution buffer or the reconstitution buffer) might have got contaminated. In fact, we have seen that keeping the unpurified PCR product in the same PCR tubes for longer duration is better than keeping the purified amplicons. This may be due to the fact that DNAses, if any, get inactivated by repeated incubation at 94-95C through the PCR cycles.
if necessary (in case of multiple products, primer dimers etc.) we prefer to purify the amplicons just before setting up the cycle sequencing reactions.
How long has you had stored the amplicons?
What characteristics have your target secuence and primers?
Maybe how many minutes are you spin-drying when you purifies your amplicons?
What kind of master mix did you use?
Maybe if you tell us about your methods something could be fail...