Dear All
I have done gel extraction my PCR product (2300bp), but the final I lose all my DNA. Here my following step. Ethanol precipitation (using glycogen, Na-OCH, ethanol) My PCR product 50 ng/µl with volume 50 µl then add TE 10 µl, and continue running the gel 3 hours 50 volt using concentration gel 1% when I cut the gel it is very hard to find band my DNA and when I continue purification gel extraction I did not find any band. It is means I lose all my DNA. It confused me. because when I tryed gel extraction my PCR product without purification before, I succeed got the band.( I dilute 10 µl running gel agarose 40 minute 50volt). Please give suggestion. Thanks very much for your attention. I really need direction and suggestion about that.
Best Regards,
Dewi Yuliana
Not sure why are EtoH precipitating before running gel? Depending on the method used, you loose a quite a bit of DNA during extraction from gel. You could enrich PCR product using the 1st PCR product as template, then run gel. You can use multiple lanes if too much volume.
Dear Dewi
it is not suggested to add any thing to your pcr product, just run all your pcr product on 1% agarose gel( based on your dna fragment lenght), then cut the gel inclouding your fragment and do gel extraction with any gel extract kit that you have. After all, if you would like to have more purified dna you can do ethanol precipitation using Amunim acetate. But I myself usually dont do ethanol precipitation.
even if you dont use kits to extract your dna, and you would like to use old methods you have to first run your pcr products on a gel without adding any thing eles to that.
how long was your gel?..did you monitor the loading dye migration?..3 hours electrophoresis is too long if you just want to see the band and purified it..
you said at first you use 45min 50V and succeded in getting the band. so why you change it to 3hours 50V?
I think like above comment:
1. Generate more concentrated PCR product (So, not necessary to EtOH precipitation, and easy to see band on gel electrophoresis)
2. For size around 2 kb, I normally run on 0.8-1% agarose in TBE buffer with 100 V, 30 min is enough (in my case)
3. Sometime Ethidium bromine or something that use for visualize DNA band is decrease because of use for staining many time. You should prepare new staining solution.
4. Use the right PCR purification band. I compare columns from two different company. Then, the yield of two company are apparently different.
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