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.

Thanks, but I wanted to know the specific reason behind your choice, so that I can compare the two methods. It'll be good if you share the pros and cons.

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.

Syed,

Do you think the yield is greater in phenol/choloroform purification than gel excision?

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.

I have never used restriction free cloning, but thanks for the advice. I have heard it is a good method!