Northern Transfer buffer?

Chris Skory skorycd at ncaur1.ncaur.gov
Wed Nov 15 09:58:10 EST 1995


Unfortunately, I don't have the references for the protocol that I use, but I remember 
that they came out of the product literature that a couple of  mol. bio. companies 
sent.  Basically, I run the formaldehyde gel with ethidium bromide added to the 
samples and then photograph before transfer. You do not need to run duplicate lanes 
for photographing since this does not affect transfer.  No other step is needed to 
prep the gel other than briefly rinsing with phosphate buffer.  Then use downward 
alkaline transfer as described below.  I just love this protocol because it always 
seems to work well.

Christopher D. Skory, Ph.D. 			National Center for Agricultural	
 
Research Microbiologist 			Utilization Research, USDA-ARS
						Fermentation Biochemistry Unit 
E-mail:  skorycd at ncaur1.ncaur.gov		Peoria, Illinois 61571-3902       

*********************************************************
Dry 10 micrograms of RNA in speed-vac.
	Resuspend in:
		1.5 ul 10x MOPS
		7.5 ul formamide
		1 ul formaldehyde
		3.75 ul ethidium bromide (0.2 mg/ml)
		1.25 ul water

Heat 65 deg, 15 min, then ice
Add 1/10 vol loading buffer (50% glycerol, 1 mM EDTA, 0.25% BPB & XC)
Load samples on 1% Formaldehyde gel

	Gel:	0.7 g agarose
		7 ml MOPS
		51 ml DEPC treated water
		        	Dissolve agarose, cool to 65 deg then add
 			12.25 ml formaldehyde

Photograph gel with ruler next to ladder then transfer RNA using downward alkaline 
transfer (as described in Current Protocols)  with the following buffer for 
Boehringer's Genius detection system using charged nylon:

	3 M NaCl (DEPC treated)
	8 mM NaOH			|   added after
	2 mM Na-lauryl sarkosine	|   autoclaving

I actually prefer to use two buffer reservoirs for the wick so that buffer transfer is 
a litter more uniform.  I don't have any data to support that it works any better, but 
I feel more confident.
    e.g.:)

              wick
         ______________    
   |___/_|  #######   |_\___|
   buffer    gel/         buffer
   tank    paper towels   tank

(This step only takes 1 hr.)
After transfer, rinse membrane with phosphate buffer and dry at 120 deg for 15 min.  
That's all there is to it!  

********************************************************

In article <upperman.816035285 at njmsa>, upperman at njmsa.UMDNJ.EDU says...
>
>
>i have a simple question.  someone on our floor claims that you can use 
>1X TAE as a transfer buffer for northern blot transfer in place of 10 or 
>20X SSC.  i say no...they say yes...comments?




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