while working with low concentrated mammalian genomic DNA, can we increase the restriction digestion reaction volume to 200 ul to accommodate the required amount of mammalian genomic DNA for realizing a visible signal with DIG labelled probes?
I think the volume for doing the restriction digest is not important. The issue is how to load it on the gel, since 200ul is a lot for one lane on a gel. But if you precipitate or otherwise concentrate the DNA after digestion and resuspend in a small volume for loading, then you can do your digestion in any volume you like.
Thank you Dr. Micheal for your valuable suggestion. In the process of precipitation/concentration, will there not be a loss in the yield?
Will increasing the width of the slot (to accommodate large volume) by combining two or three teeth has any effect on the resolution of fragments or signal? what is the ideal gel thickness for efficient transfer of DNA onto membrane?
If you add carrier like tRNA or glycogen or non-homologous sheared DNA you can get high efficiency precipitation.
Using a larger well will dilute the band by dispersing it over a larger area.
Michael J. Benedik what are the chances of renaturation of the probe during hybridization?
Michael J. Benedik Sir, When we try to precipitate the restriction digested products, will the salts not interfere the transfer of DNA onto the nylon membrane?
Among 20X SSC and 10X SSC, which is a better transfer buffer?
Thank you.
No, the salts don’t come along after ethanol precipitation and even if they did the next step is running a gel and they would be lost there.
Michael J. Benedik Sir, as suggested, I have precipitated the restriction digested products, and tried to transfer the precipitated fragments onto positively charged nylon membrane manually using 10 X SSC as the transfer buffer (for around 18 hrs).
I have noticed a lot of DNA in the gel post transfer to the membrane. In lanes marked 2,3 and 4 restriction digested products were precipitated and loaded, where as in lane 4 restriction product was loaded directly by combing two wells.
Could you suggest me ways to improve the transfer of DNA onto the membrane, so that enough DNA would be there on the membrane to give signal post washes and detection.
Thank you.
I have not done a southern blot in a very very long time, so I'm not the best person to ask about how to improve transfer. You might repost that precise question on researchgate and you are likely to get some answers.
The two things I do remember is that electrophoretic transfer to membranes is much more efficient and secondly to be sure that there is not buffer short circuit, that the only route for the buffer is through the gel and there is no contact between the buffer and the pads above the membrane except through the gel (not at the edges).
Michael J. Benedik Okay SIr thank you, I have posted it again, let me try my best.
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