I extracted rice leaves samples using CTAB (organic) protocol. The lysis step takes 3 hours at 65C. Purification is by chloroform:isoamyl alcohol (24:1), 10mins of mixing and 10mins of centrifugation. Then precipitation in isopropanol (-20C, overnight) then the precipitate is dissolved in TE buffer. RNAse A and proteinase K treatment (1hour at 37C) followed. Then precipitation again with 4M sodium acetate+ absolute ethanol (-80C, overnight), then 70% EtOH washing of the pellet. The final precipitate is then dissolved with RNAse-free water. When I ran the samples in a 1% agarose gel, 100volts for 35mins, these smears were observed. These samples are for Genotyping-by-Sequencing but it is required for such procedure that there should be no smearing on agarose gel. Any ideas?
Hi, maybe you try to wash it twice with 70% EtOH (but of course you will loose some DNA...).
Cheers, Nadine
Hi Nadine,
Yes I tried precipitating it twice and it worked. However, the yield reduced to almost half...
Thank you :-)
Shiela
The samples may be having too much salt due to precipitation with 3M sodium acetate, so give washes with 70% ethanol thoroughly. Also, take care when collecting the aqueous phase during the extraction process.
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