Is there any time limit to do sequencing after the nested PCR?
Amplified DNA is very stable. I would recommend to store the DNA at 4°C for a couple of days - freeze your samples for a long term storage (-20° is ok).
A very important point is the purification! For a good sequencing quality you have to remove the PCR primers!
if your amplicons are well preserved or stored at -80 c or liquid nitrogen fine.
Amplified DNA is extremely stable. If you store your amplicons at -20C you should be able to sequence it several years after.
I have obtained satisfactory results after a -20C storage for 10 months.
I agree with Artur on that what matters is the quality. If you can still see the bands in a gel, that should be ok.
I don't think nested PCR product is any less stable than a normal PCR product. I would purify it out of the PCR reaction mix, by gel, and then store at -20℃, or even 4℃. There must be a good reason why you are doing nested PCR, so you will want to make sure you are keeping the right target amplicon.
If you run your own sequencing reaction before sending to sequencing facility, the dried product is very stable at -20℃, but keep it out of light.
As every one else has said correct storage of the product is very important. You can purify your products and than save them at -80. It has worked for me.
Amplified DNA is very stable. I would recommend to store the DNA at 4°C for a couple of days - freeze your samples for a long term storage (-20° is ok).
A very important point is the purification! For a good sequencing quality you have to remove the PCR primers!
Georges Wigger's comment is quite correct. In addition to this - it whoud be better to store only purified PCR products (dissolved/eluted in HPLS water not less than 18 MOm) at +4C if you want to sequence them for a month. For long period storage you can use -20C, but be careful with quantity of freezing-thawing cycles: DNA samples longer than 1000 bp can partially degrade after 3-4 times of such cycles!
Hello
If you store your PCR products at -20 deg for a long term you can use .
Good Luck
If preventive measures have been pondered all should be ok. Amplified DNA is very stable. More relevant, is evaluate if the sample in the end are well amplified and the DNA not suffering degradation.
If you not plan using immediately the amplicons obtained (only in the near days) you should store them at +4ºC ( and if you have sufficient volume of amplicons store half in aliquotes at -80ºC for later experiments or validation). For some mid/long term storage go for -20ºC or even -80ºC having caution with the medium used and should not forget to use one crioprotector.
It is still working on my DNA sample(PCR product) when it has been stored at 4ºC refrigerator for 1 month at least. Sequencing result is quite good to me, but it depends on your DNA quality also. The other way you might do by cloning you DNA into plasmid ( pGEM-T easy vector for example), which makes you DNA more stable
Storage of your amplified DNA at -20°C is fine and allows sequencing even after years. To avoid confusion about the quality of the sample if sequencing is scheduled for later times by another person, which might not be familar with the protocol, the sample should be purified (primer removal etc.) to be ready to use.
Storage of your amplicon at -20°C is fine and you can do sequencing even after years, but to check on gel before send sequencing.........
I do bisulfite sequencing and store PCR products in -20C for many months. I use XP beads (Beckman Coulter) to clean-up the samples.
no, there is no limitation. as long as there is no contamination it can be stable for few weeks in 4C and in -20 for many years. but to have better result from sequencing it would much better if you clean up your PCR product by for example AMPure XP bead and then store it.
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