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djvan's Content

There have been 3 items by djvan (Search limited from 25-October 19)


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#178445 Gel Electrophoresis: Gel Poured With Water

Posted by djvan on 30 July 2016 - 09:12 AM in General Lab Techniques

Thanks Bob.  Electricity/current never was my forté.




#178441 Gel Electrophoresis: Gel Poured With Water

Posted by djvan on 29 July 2016 - 01:04 PM in General Lab Techniques

Hello,

 

I'm in the process of training a recent college grad, and they accidentally made the mistake of pouring an agarose gel with water instead of buffer.  As expected, their gel overheated and they lost their DNA.

 

I started reading online as to why this happens, and couldn't find any scientific explanation.  So, can anyone explain why a gel cast with water, and run in a chamber with 1X TBE, conducts electricity better (I assume this is where the heat is coming from) than a gel cast with buffer?

 

Thanks!




#177998 UV-Imaging Chambers: Oversaturation from many gel lanes?

Posted by djvan on 09 April 2016 - 10:40 AM in General Lab Techniques

Hi All,

 

I've been running some massive DNA-agarose gels in a screening application downstream of PCR.  The gels contain 4 combs of 50 lanes each.  The center of the lanes within a comb are separated by about 5mm.  I cut the gel prior to imaging, so that only 50 lanes are imaged at a time.

 

I think I'm running into some problems with these large gels in relation to the UV signal.  I think I might not be detecting weak (low concentration) DNA bands on these gels.  Can bright DNA bands in adjacent lanes 'mask' weak DNA bands lanes?  The camera needs to be zoomed-out pretty far to encompass all of the lanes, and I think I might be losing sensitivity?  I noticed if I crank up the exposure, weaker bands do appear, but the other, stronger bands, become oversaturated as does my DNA ladder.

 

I could load more of these weaker samples, but the issue is I will not know which lanes need to have more loaded until after I run the first gel.  It would be ideal to only have to run the gel once, as this is a screening process and quite laborious.

 

Thanks for any help!





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