pcr product purification - (Aug/28/2007 )

hi
after pcr which way is better to purify the pcr product
with phenol:chloroform:isoamyl alcohol (25:24:1) ratio
or
with gel extraction kits

which way is efficient for removal of polymerases and primers and all impurities

please suggest
thank you

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i don't do phenol chloroform... because it's not as done in my lab when i arrived....

I BAN gel purification for pcr i want to digetst and ligate, and do column/or matrix kit directly on pcr mix.
Nice

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There are PCR purification kits also, with just a colonn, if you just want to purify your product from primers and so. Much easier than having to do a gelpurification.

I have never done a phenol chloroform purification, I have only used QIAquick PCR purification kit and their gel purification kit.

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hi thank you
I don't have pcr purification kit
that why i used to go phenol for removal of enzyme then gel extraction to remove primers
is gel extraction alone can purify everything
please suggest
thank u

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normally not. you can get rid of most of the nucleotides but for proteins there remain few on eluate.

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Just do a standard EtOH precipitation, but if the product is less than 200 bp I'd suggest ammonium acetate rather than sodium acetate. Add 1/2 vol 7.5 M, pH7.5, to DNA and 2 vol 95% EtOH, and precipitate @ -70C for 30-60 min if [DNA] > 50ug/ml; otherwise -20C overnight.

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hi thank you
i have similar problem while digestion of vector and insert
here i am digesting vector with Nde1 and then i am doing gel extraction of linearised vector
then proceeding to second digestion with BamH1
in this case i am getting more loss of DNA
please suggest me if any alternative for purifying after first digestion
is that dna can be purified compltely in gel extraction kit
is taht gel extraction cal eliminate all of the enzymes from DNA

plaese give suggestions

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Do a standard EtOH precipitation with Na acetate.

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