I am isolating plasmid DNA using my own buffers and found the OD 260/280-1.93 and 260/230-2.19 the concentration is also as good as the commercial kit available in market. I have used 15ug/mL RNAse in 50mM Tris +10mMEDTA for re-suspension the bacterial pellet.
Lysis was carried in 200mM NaOH + 1% SDS and finally neutralize in 4.2M Gu-HCl and 0.9M CH3CooK (pH-4.8) and used commercial columns for binding DNA. The columns were washed with 80% EtOL twice and dried as normal protocol and finally the DNA was eluted in 65ul EB. Everything is fine but when I check the quality of DNA on the gel I noticed a little background of DNA smear in each lane. Please view the picture in attachment.
Last two right lanes do not have the smear in lane in which I have used commercial buffers.
Please suggest how to avoid the smear?
Hi,
I think it's not too bad. Maybe due to overload of your sample.
Hi,
please compare your purification protocol with the one described in:
M.M. Diogo, J.A. Queiroz, G.A. Monteiro, S.A. Martins, G.N.M. Ferreira, D.M.F.
535 Prazeres, Biotechnol. Bioeng. 68 (2000) 576. Instead of using the resin they refer, I usuallly use Phenyl sepharose fast flow. The results are usually very good in terms of purity (kit comparison) and yield (pDNA concentrations around 300 ug/ml)
Hope that helped.
Gabriela
GuHCl causes precipitation with SDS, so the smear may be due to that. I know I often have smear PROTEIN gels when using GuHCL with SDS based PAGE. Might be your issue. You could theoretically substitute a higher molarity of Urea instead of GuHCL, maybe 5-6M Urea.
Hi,
I think it's not too bad. Maybe due to overload of your sample.
I agree with Hetty that it may simply be a factor of the quantity of DNA loaded on the gel. They look pretty clean to me. The important question is what you will be using the DNA for and how pure do you therefore need to get the DNA?
Dear Hetty
It seems it is little smear, i tried to digest this DNA with various RE and amount of smear increased, now i assuming it b/c of DNase contamination which increase its activity in RE buffers. If you want i can mail you the picture. I am thinking to use proteinase K starting from resuspension buffer. Proteinase k work even in presence of GuHcl and enhance the activiuty in Sds and NaOH. I think commercial kit manufacturer use this while some only mention about it. Hoping to get suggestion from you.
Vishal
HI HETTY
these are the enzyme and buffer from NEB and for double check I have used the DNA FROM other preparation and these are clean and no smear after digestion if it is from RE AND Buffer it may also show the smear in that DNA also.
Vishal
Dear Vishal Gupta,
on my opinion, I suggest that there is no smear in the gel that you sent. This is normal background noise in migration due to overloading and matrix effects - which a suggestion is that in part it can be solved by cooling the gel (put it in refrigirator) and slowing down the migration - use lower voltages (also helps to cool down gel), besides lower amount sample.
Regarding smear, I recommend our article on the subject (includes short presentation): http://www.sciencedirect.com/science/article/pii/S0167701213002467
Best regards,
Dr. Jan Zrimec
I have resolved the smear issue by increasing potassium acetate in binding buffer.
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