I have extracted DNA from rice leaves using the modified CTAB DNA isolation protocol, Nano drop reading is showing 1.8 to 2.00 at 260/280 ratio and the concentration is above 1800 nanogram per microlitre. After RNase treatment, when I check the quality of extracted DNA by means of agarose gel electrophoresis I have long band smeared that is lower the DNA band.
I understand this is a sheared DNA,
I want to know is it usable for further PCR amplification works?
although you have smearing there is good amount of intact DNA ,I think you can do PCR !!
Thank you Zx Ching and Abrar Jamous for your suggestion, I will surely try that.
Hi, Dalia Sabrei, I used Ethidium Bromide for the gel.
Hi Deepak, to me it does not look much shearing or smearing, as bulk of your DNA still visible and trapped near the wells. Since you have very good amount of DNA so overloading of wells with such huge amount may also result in smearing. Also using dirty gel equipment, having above 10 kb-sized DNA molecules, using of excess gel running buffer (above 5 mm over the gel surface), increased voltage, old buffers, these all in fact slows the DNA movement on gel and may result in smearing. Only the fully solubilized DNA runs properly on gel.
Since your OD260/280s are excellent and you have plenty of DNA in terms of concentration so you must proceed with confidence for the PCR. Also it is not imperative to perform RNAse treatment, as RNA won't have much effect due to the staging & cycling conditions of PCR. It will get rid off from the reaction in the first phase of PCR, which is denaturation.
Regards....
Hi Jawwad Ahmad,
Thank you for taking time in responding to my questions. Those are some important tips you mentioned. from now on I will take care of that too in future.
Thank you once again.
many regards
deepak
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