I will be sending my samples for next generation sequencing in a few weeks time. Is it ok to purify samples now and keep them at -20 or better to purify few days before sending for sequencing?
Better to purify sample right after PCR to avoid any residual enzyme activity.
Better to purify sample right after PCR to avoid any residual enzyme activity.
I agree with Pranali, better clean them right after and avoid freezing/thawing as much as possible.
I agree with them, The best think it´s to purify them now and then you have the samples prepared to send and to sequence. They will be already frozen and prepared for that moment and you avoid to freeze and thaw.
I agree to the above comments. Purify and send for sequencing ASAP is advantageous to avoid possible mutations, and other enzymatic chemical reaction as mentioned above(Pranalli Deore).
Check this out if it helps: http://seqcore.brcf.med.umich.edu/doc/dnaseq/pcr.html
Good luck , Sathiya
Agree with the above. Its always best to purify samples after PCR and store for future use. As Monika stated above avoid freeze thaws as best you can.
Purified porduct is more stable. I do this with my sample. I advice you to keep a store part from the purified product for future use.
furthermore… if you purified right away, you won't have to unfroze them one more time to purify them if needed :p
You can store it in TE buffer and I also experienced that the products are more stable in gel. so you can store directly (note the weight at that time) the cutting of gel containing DNA and can elute it when needed.
purification of the PCR product before sending them for sequencing give high reliability.
I purify my PCR products right away no matter what I do with them downstream for eg cloning, sequencing, anything!!! I think it is always better to remove any kind of DNases. As you see that is a unanimous suggestion.
It is better to purify samples after PCR and keep them in liquid nitrogene or at -20.
I always purify PCRs after checking them by gel electrophoresis. Certain polymerases with proof-reading activity may also degrade your PCR product (e.g. Pfu polymerase). This is not the case of Taq polymerases, however, it is always better to purify PCR products before storage. Column based purification kits work perfectly well. Remember that, if you are going to use them for sequencing, PCR product has to be eluted in water or Tris buffer without EDTA!
Its advice to purify your PCR sample straight away, it increases your sample stability and avoid any end point degradation. For sequencing we need and high quality sample for better result.
The purified PCR product is more stable, since it has removed the enzyme and other ions in the buffer.
For NGS level, It is better to purify samples after PCR and keep them in liquid nitrogene or at -20. based on your available facility.
Hi Sathiya!
My advice is to purify the amplicons and afterwards keep them at - 20C. Purifying the PCR products will give you a better sequencing result.
Good luck!
Thank you all for the recommendation. I will purify the samples and store at -20.
- Badges
- Science method