alkaline transfer protocol

bckraev bckraev at
Fri Jan 13 13:55:55 EST 1995

Hi, Randall. Here is my version. If you need more details, feel free to ask
again. Have a nice weekend. Sasha

Rapid Downward Alkaline Transfer of DNA from Agarose
 Gels to a Positively Charged Nylon Membrane

1. The gel is made of 50 ml  1% agarose on TAE buffer in a microtiter plate 
(80x125 mm ) and has a thickness of about 3 mm. The gel is typically run  
for 45 min at 
ca 8 V/cm until the bromophenol blue reaches bottom ( shortest visible 
fragments about 
500 bp ).

2. The gel is stained in 500 ml of 0.5 ug/ml EtBr in TAE by shaking for 
10-20 minutes,  
then the staining solution is replaced with 500 ml of water and a picture 
is taken  from the 
gel under a UV light source.

3. The gel is shaken  for 10 minutes in 400 ml of 0.25 N HCl, prepared from 
a standard 
solution ( e.g. Titrisol, Merck ). The bromophenol blue band changes colour 
to yellow. 
The acid solution is replaced by 500 ml of 0.5 M alkali solution 
(10gNaOH pellets /500 ml ). The shaking continued until the bromophenol blue 
band turns blue again ( about 10 min ). 

4. The tank with the alkali solution ( with the gel still floating there ) 
is used to wet three 
pieces of Whatmann 3MM paper cut exactly to the size of the gel 
( 80x125 mm ) . The 
wet paper stack is placed on a glass plate, which is only slightly larger 
than the gel, the 
gel is carefully inverted and laid on this stack. If the gel does not seem 
to be completely 
flat,  cut about 3 mm from the edges.  Then a dry nylon membrane  
( 100x140 mm from 
Boehringer Mannheim ) is laid on the gel , followed by another wet piece of 
paper. A  10-ml pipet is rolled over the whole stack,  with moderate pressure 
applied, to 
squeeze all air bubbles and excess alkali solution.   A paper towel stack, of 
the size larger 
then the sandwich by about 1 cm on each side, and about 2 cm thick, is then 
put on the 
sandwich , followed by a second glass plate. The whole construction is  
carefully inverted 
and left on the bench for about two hours ( or overnight, if convenient ). 

5. The sandwich is carefully disassembled and the membrane is peeled off the 
gel, which 
should be compressed to a thickness of about 1-2 mm . A piece of Whatmann 3MM 
wetted with a few milliliters of 3 M sodium acetate,pH 5.5  and the wet 
membrane is 
placed on this paper for 1-2 min to neutralise the alkali. Immediately after 
that the 
membrane is placed on taut Saran Wrap, DNA side down, and transferred onto 
surface of a short wavelength UV transilluminator. The membrane is irradiated 
for 2-5 
min, transferred into about 50 ml of 0.05 M phosphate buffer and shaken 
briefly by hand. 
The membrane can be dried at this stage and stored indefinitely, or immediately 
hybridized to a probe.

Stock solutions required:

1. TAE agarose buffer, 50X
2. Ethidium Bromide, 10 mg/ml
3. 1 M HCl, standard solution
4. 0.5 M NaOH solution, freshly prepared
5. 3 M sodium acetate, pH 5.5 
6. 0.5 M sodium phosphate solution ( 89.5 g disodium hydrogen phosphate and 4 ml of  
  85% phosphoric acid to 500 ml  water ). 

(end of file)

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