According to protocols I found from Nature (Heckman et al 2007) and Cold Springs Harbour (Forloni et al 2018), the PCR products must be purified using gel electrophoresis and then a QIAquick gel extraction kit or a similar product. According to a senior person in my lab, we usually use ethanol precipitation for PCR products because the gel method results in loss of a lot of the product. Is this a question of quality over quantity?
Also, is there a difference between the QIAquick gel extraction kit and the QIAquick PCR Purification kit, and which is better?
Hi Melissa,
I have used the QIAquick gel extraction kit for years and now have stepped over to purification by AMPure paramagnetic beads that bind to pcr amplicons of 100bp and larger. So by a few washing steps you will quickly remove excess primers, salts, enzymes etc. The gel method works perfect , but you will indeed lose at least 50% of your product. Sometimes when you have weak bands, this will result in too low concentrations for final library prep. So I just check on gel, but purify with AMPure. Its not only fast, but in time you will save alot on kits. In 30 minutes you can easily purify a complete 96well pcr plate. I only use ethanol precipitation when my concentrations are too low ( measure with Qubit) , lets say under 0,7ng/µl.
greetings
Gerrit
In my opinion after electrophoreis for check ... the PCR product in PCR tube send it for sequencing without extraction from gel ... just the PCR product after detect by gel electrophoreis.
There are two ways to purify your PCR product:
1) After PCR, try run a bit of your PCR product on a gel, if your pcr product only yield one band, then, you can just purify the balance of the pcr product straight away using QIAquick PCR Purification kit without the need to gel those PCR product. QIAquick PCR Purification kit is used only when you purify from pcr product without gelling.
2) After PCR, try run a bit of your PCR product on a gel, if your PCR product gives multiple bands whereby only one band is your desired product, the you have to gel all the PCR product, cut the desired band on the gel and purify it using QIAquick gel extraction kit. QIAquick gel extraction kit is used when you gel your PCR product.
We directly use the TIANGEN Kit to extract DNA for SOE PCR. So far, never fail
We also use purify DNA after electrophoreses only if spectrometry analisys showes that it's not clean as ie should be, otherwise it goes directly to sequencing
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