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How do I determine sizes/lengths on Northern blots?

How do I determine sizes/lengths on Northern blots? - Protocols and Methods Forum

How do I determine sizes/lengths on Northern blots? - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 07-16-2003, 04:56 PM
Peter Frank
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Default How do I determine sizes/lengths on Northern blots?



Which is the best method to determine sizes/lengths of RNA bands on
Northern blots? After all, lengths of RNA marker bands cannot be
"seen" directly on the blot.

I can think of two methods:
* The RNA marker lane is cut off the gel before blotting and stained
with EtBr and photographed. The relative migration distances are used
to determine lengths on the blots.
* In "Molecular Cloning" (Maniatis et al.) they say that RNA can be
stained on blots by using a special methylene blue solution. So, the
RNA marker lane could be blotted together with the rest of the gel and
then the RNA marker lane could be cut off to be stained with methylene
blue.

Which one is more commonly used? Oddly enough, the papers in which
Northern blots were made use of do not mention how the RNA sizes of
bands on a Northern blot was determined. They just say that certain
RNA markers and/or 18S- as well as 28S-rRNA were used as size markers.
That is why I would like to know how it is usually done.

Peter
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  #2  
Old 07-16-2003, 05:11 PM
Gys de Jongh
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Default How do I determine sizes/lengths on Northern blots?


"Peter Frank" <[Only registered users see links. ]> wrote in message
news:[Only registered users see links. ]...

Hi,
methylene blue (do NOT use bromophenol blue!!) is positively charged. It colors
any neg macro molecule , like RNA . You can just dip your BLOT in a methylene
blue solution and take a photograph with your standard equipment using daylight
instead of UV. The washing buffers in the protocol contain SDS which is neg
charged. This soap will remove the pos charged methylene blue very effectively.
So no cutting.
Works great for me.

--
Gys


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  #3  
Old 07-16-2003, 07:02 PM
Michael L. Sullivan
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Default How do I determine sizes/lengths on Northern blots?

>Which is the best method to determine sizes/lengths of RNA bands on

I usually run my Northerns with EtBr in the gel, so it's already
stained after the run. I then poke the gel with a pasture pipette at
the position of each marker band. Following blotting the gel (and
before separating the gel and the membrane, you can flip the
gel/membrane combo so the gel side is up and run your finger over the
gel surface which will reveal the places you poked the gel. You can
use a pencil to mark the position of each marker hole you made in the
gel onto the membrane. Post hybridization and imaging, you can line
up the blot with the film or image and estimate the sizes of your
bands of interest.

Mike
--

Michael L. Sullivan, Ph.D
Research Plant Molecular
Geneticist
U.S. Dairy Forage Research Center
1925 Linden Drive West
Madison WI, 53706

(608) 264-5397 Phone
(608) 264-5147 Fax
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  #4  
Old 07-17-2003, 05:14 AM
EK
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Default How do I determine sizes/lengths on Northern blots?


"Tom Anderson" <[Only registered users see links. ].ac.uk> wrote in message
news:[Only registered users see links. ].ac.uk...

Even better, Tom. Let's use pasture pipette in Cow protocol !


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  #5  
Old 07-17-2003, 04:19 PM
Dr. Hiranya S. Roychowdhury
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Default How do I determine sizes/lengths on Northern blots?

Both methods are just fine. Choose the one that you can handle. However, this
is what is easiest:

Run the gel with the markers and then, while visualizing on the
transilluminator, make incisions on the marker bands. You do not have to stab
the bands all the way. Transfer as usual and before peel off the membrane,
turn the gel over and, the same way you mark the lane wells - mark the marker
bands on the membrane, through the gashes, with a soft "lead" pencil (HB or
2B). You then have a permanent record of right on the norther blot. Since a
properly handled northern blot is usually an asset for the entire lab for years
to come, this method obviates the task of checking the original investigator's
notebook.

Hiranya



Quoting Peter Frank <[Only registered users see links. ]>:



--
Hiranya S. Roychowdhury, Ph.D.
Coll. Asst. Professor,
Molecular Biology,
Dept. of Chemistry & Biochemistry
Rm# 336, Chemistry Bldg.; MSC 3MLS
New Mexico State University
Las Cruces, NM 88003
---
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