In terms of loss, quality and successful cloning, which method is better to use for purifying the PCR amplified insert and the double digested insert, Phenol-Chloroform -Isoamyl alcohol extraction or gel purification? Please let me know the pros and cons of both the technique to help me decide.
Ofcourse Phenol/Chloroform/Isoamyl alcohol followed by EthOH precipitation. However, if the DNA fragment is contaminated with undesired fragments then there is no other option but gel purification.
Purity and yield of DNA are not as good from silica column -based kits. On the other hand, DNA purification using kits is relatively faster.
Hi there,
Gel extraction+silica gel purification is definitely the best method even if yield is not great. For PCR amplified product it results in total elimination of the DNA template and for digested product it results in elimination of digest by-products which may interfere with downstream applications and which may copurify when performing phenol extraction and ethanol precipitation.
Yes, it is always greater. However, like I said before, if the PCR reaction contains additional amplicons (judged by agarose gel electrophoresis) then one has to gel purify the desired product.
We should keep the two situations separate; PCR -amplified products, if not contaminated with undesired amplicons, can be recovered in higher yields using the PCI/EthOH precipitation method. Restricted DNA fragments need to be purified from the gel.
For my cloning experiments, I heavily rely on whole plasmid PCR (to introduce desired restriction enzyme sites or RBS etc.). After PCR, I add DpnI into PCR reaction to get rid of template DNA and purify the amplified DNA using PCI/EthOH. I have found PCI/EthOH purified DNA to be restricted very well compared to column -purified DNA.
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