Hi!
Is attached a picture of the agarose 1% gel of my DNA extraction (fungi, Colletotrichum).
The bands are smeared and appeared a second band next to the well.
I used CTAB 2% as extraction buffer (980 uL of buffer + 20 uL of Proteinase K, overnight at 50 C; Proteinase K inativation at 95 C for 10 min; added 2 uL of RNAse, 1 hour at 37 C). The aqueous phase was separated by 1 wash with 700 uL of CIA, and another wash with 700 uL of CIA and 200 uL of CTAB 10%(after the second wash, almost didn't see the protein layer). The DNA was precipitated with isopropanol (overnight) and pellet was washed with 70% ethanol. The pellet was diluted in 60 uL of TE (the pellet dissolved fast).
The DNA concentration was between 200 -- 600 ng/uL. The 260/280 and 260/230 ratio were between 1.7 -- 2.0.
I used 2 uL of sample and 2 uL of xylen cianol + gelred to run the samples in the agarose 1% gel. I ran at 60V for 2 hours.
About the smeared bans, did I use a small amount of sample to load the well?
And what`s this second band band close to the well?
The DNA will be used to genomic library. May this second band cause any problem?
Thank you!
Hi there,
It is typical of genomic extraction of fungal DNA, not to worry though! The "band" in the well is probably some protein/sugar/DNA leftovers not completely removed from the solution that catch some EtBr and the smear for the DNA band is a small degradation of the DNA... but so far your genomic DNA looks perfectly fine to me, and it will be just fine for genomic library ;-) ! Good work and good luck for the future !
Mat
Hi willie
May be i am expecting good band in higher % that is 1.2%
Thank you
Hi there,
It is typical of genomic extraction of fungal DNA, not to worry though! The "band" in the well is probably some protein/sugar/DNA leftovers not completely removed from the solution that catch some EtBr and the smear for the DNA band is a small degradation of the DNA... but so far your genomic DNA looks perfectly fine to me, and it will be just fine for genomic library ;-) ! Good work and good luck for the future !
Mat
The eighth sample is the best, you know the reason in your manupulate?
Mathias is right and you should not worry about the extraction, it is a typical one. In addition to Mathias suggestion, other possibility to explain the material just below the well is that it can be DNA that is not properly suspended in the buffer/water. When you overdried your samples, it is difficult to suspend all the material in a water-based solution, and therefore does not run into the agarose gel. Solution, add more water/buffer and mix well!
I used to do many Magnaporthe DNA preps and I always would have contaminants like polysaccharides. It is extremely difficult to get rid of them because they always in the same fraction as DNA. The only way which worked for me is slow gradual precipitation with NaAc and ethanol. In any case, your preps look great and, unless you need high purity DNA for whatever reason, your preps are good for majority applications. Good Luck!
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