I was using the promega DNA extraction kit to extract my DNA from whole blood and I used to resuspend DNA in RNAse free water and the PCR was working fine. Lately I used the genejet DNA extraction kit and eluted my DNA in like 400 ul TE buffer (10 mmol tris-0.5 mm EDTA) which made DNA very dilute (14 ng/ul). I tried using 10 ul for PCR but it didn't work. Could TE edta be hindering reaction one more Question, can I reuse spin column and reelute DNA in smaller volume of water?
PCR buffer contains ~ 1.8 mM Mg++. EDTA chelates 4 Mg++ ions per molecule. With 10 µl TE in a 50 µl reaction your final [EDTA] is 0.1 mM. This will drop your final [Mg++] to ~1.4 mM which could certainly have an effect. Yes, you can re-use columns as long as the DNA is the same type as used previously.
in addition to Marks comment on the Mg con, you may consider reducing the amount of buffer to concentrate your DNA . another option would be to elute in RNAse and DNA se free water.
the re-use of column is only for the exact same sample. I would not recommend it for different samples.
I intend to use it for same sameple but in DNAse free water.Any specific protocol for that?
there is not specific protocol just use a lower volume of water (100ul) to increase your DNA yield.
I don't recommend using water because depending on how long it has been sitting around, it could have become acidified and depurinate the DNA. Instead use 10 mM Tris-HCl, pH8.0. There shouldn't be any nucleases in column-purified DNA so omission of EDTA should be fine. Alternatively, use T0.1E (i.e. TE with 0.1mM EDTA instead of 1 mM).
If DNA is dissolved in TE buffer,Can i reload it and elute in smaller volume?
I don't know if that's even possible as it is already dissolved in eluate.
Yes you can but you will need to add fresh gel dissolution buffer, or binding buffer ( if the kit contains it) before loading the column. The DNA will not stick to the column matrix unless it has the requisite salt in co-solution.
DO NOT follow Hemin's advice. If you add isopropanol without salt, you will lose 95% of your DNA!
DNA extraction column may be reused after treating with 1M HCl overnight: http://www.ncbi.nlm.nih.gov/pubmed/17373483
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