SEMI-DRY BLOTTING

masklinkem at gemini.ldc.lu.se masklinkem at gemini.ldc.lu.se
Tue Apr 28 11:15:28 EST 1992


A lot of people have requested additional information about 
our experience with SEMI-DRY blotting, so I will post it for general viewing.

We have used the system for protein, DNA & RNA with
satisfaction.We use the SEMI-DRY blotter from 
KEM-EN-TEC Denmark (FAX +45 31200178)  that have graphite 
plates and built in power supply and timer. It has been used for 
years without problems. I have tryed other apparatus with 
conductive rubber, these seems to work in principle but you 
need to change the rubber frequently.
The US distributor of the apparatus is:
BIOTREK INC, 
11212 N.May Suite 405E
Oklahoma City, OK 73120, USA, 
FAX 405 751-3002, 
Phone 405 751-0008 

P.Szecsi
Dept. Clinical Chemistry
Malmo General Hospital
University of Lund Sweden
Phone +46 40336623
FAX     +46 40 929023
NET      MASKLINKEM at SELDC52.BITNET

Enclose the requested info about semi-dry blotting.


SEMI-DRY Electroblotting of DNA

Agarose Electrophoresis

Solutions

2.5xTBE buffer
	0.22M TRIS base
	0.22M boric acid
	5mM EDTA
	just pH to 8.0 with HCl

Gel-loading buffer 6x
	0.25% bromphenol blue
	0.25% xylene cyanol
	15% ficoll (type 400)
	in H2O

Fragmentation solution
	0.25N HCl

Denaturation solution
	1.0M NaCl
	0.5M NaOH

Neutralization solution
	0.5M TRIS pH 7.4
	1.5M NaCl
	
1% agarose in 2.5xTBE buffer
1.	Melt the agarose solution in a boiling water bath or in a 	microwave oven.
2.	Cool to approximal 50!C.
3.	If visualization is required ad ethitium bromide to a 	concentration of

	0.5mg/ml (from a stock solution of 10mg/ml in water, 
	stored at 4!C in a dark container).
4.	Pour the gel and allow to set (30-45 min. at room temp).
5.	Add running buffer to cover the gel to a depth of about 1mm.
6.	Samples are mixed with oading  buffer, and loaded into the gel.
7.	Electrophoresis is carried out for approximal 1 h at 100V 
	( 8x5x1 cm gel).
8.	Rinse the gel in deionized water.
9.	Fragmentate the DNA in 0.25N HCl for 5-15 min. 
10.	Rinse the gel in deionized water.
12.	Denature the DNA by immering the gel in 1M NaCl 0.5M NaOH 	twice for 15 min.
13.	Neutralize the gel in 0.5M TRIS, pH 7.4 / 1.5M NaCl twice for 
	15 min.
14.	Soak the gel in 2.5x TBE twice for 5 min.
15.	Pack a transfer sandwich in the SEMI-DRY BLOTTER II 
	avoiding airbubles according to the following bottom (plus)
	and up.

	a)  5 layers of Whatman 3MM filter paper soaked in 2.5xTBE
	b)  a wetted nylon membrane (Hybond-N, Amersham; 
	Satorius E, E & K Scientific or Nylon 66, Hoefer)
	c)  the slab gel
	d)  5 layers of Whatman 3MM filter paper soaked in 2.5xTBE


16.	Assemble the SEMI-DRY BLOTTER II and electrotransfer at 	constant current
for 15 min.
	(use approximal 3-5 mA/sq cm of gel). 
	This transfers fragments between 23kb and 0.3kb.
17.	Process the membrane according to the manufactors instruction.


Note 	If a base stable membrane is used can Step 9-13  be omitted and the
membrane can be base washed after transfer. .

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

Alternative procedure (quick and dirty)
=====================================
5xTBE buffer 
	108g TRIS base
	55g boric acid
	40ml 0.5M Na2EDTA (just to pH8.0 with NaOH)
	ad 1L H2O

Denaturation solution
	1.5M NaCl
	0.5M NaOH

Neutralization solution
	0.5M TRIS pH 7.2
	1.5M NaCl
	1mM EDTA
20xSSC
	175.3g NaCl
	88.2g sodium citrate
	in 800 ML water. Adjust pH to 7.0 with NaOH. 
	Adjust volume to 1000ML.


1.	Follow the procedure according to tep 1-7 as above 
	but use 0.5xTBE instead.
2	Soak the gel in 0.1xTBE for 30 min. at room temp.
3.	Assemble the transfer sandwich as in Step 15 in the 
	standard procedure, but with the filter papers 
	soaked in 0.1xTBE.
4.	Assemble the SEMI-DRY BLOTTER II and electrotransfer 
	at constant current for 30 min.
	(use approximal 3-5 mA/sq cm of gel)
5.	Denature the membrane in 0.5M NaOH/ 1.5M NaCl 
	for 5 min.
6.	Neutralize the membrane in 0.5M TRIS/HCl pH 7.2/
	1.5M NaCl/1mM EDTA for 5 min.
7.	Wash in 2xSSC for 2x5 min.
8.	Process the membrane according to the manufactors 
	instruction. 
**************************************************************

DNA electrophoresis in polyacrylamide
=====================================
30% acrylamide
	acrylamide			29g
	N.N+-methylene bisacrylamide	1g
	water ad				100ML

3% APS
	ammonium persulfate		0.3g
	water ad				10ML

1.	Prepare polyacrylamide solution according to TABLE and fragment size.

				Polyacrylamide gel %
					3.5	5.0	8.0	12.0	20.0
	--------------------------------------------------------------------
	30% acrylamide		11.6	16.6	26.6	40.0	66.6
	water			76.3	71.3	61.3	47.9	21.3
	3%APS		 	2.1	 2.1	 2.1	 2.1	 2.1
	before pouring add 30 ul TEMED

2.	Samples are mixed with loading  buffer, and loaded into the gel.
3.	Electrophoresis is carried out in 1xTBE for 
	approximal 2 h at 150V ( 15x15x1.5 mm 6% gel).
4.	Assemble the transfer sandwich as in Step 15 but with 
	the filter papers 
	soaked in 0.1xTBE.
5.	Assemble the SEMI-DRY BLOTTER II and electrotransfer at 	constant current for
1 hour 
	(use approximal 3-5 mA/sq cm of gel) 
	extend the time for higher concentrations of acrylamide.
6.	Proceed according to Step 3-8 in the Alternative procedure.

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

SEMI-DRY Electroblotting of RNA 
================================
	All buffers and glassware should be treated with 0..1% diethylpyrocarbonate
(DECP) for at least 12 hours at room 
temperature in order to inhibit RNAse Activity.
	 DECP inactivate the RNA by carboxymethylation, and 
DECP should be removed by autoclaving or heating to 
100!C for 15 min. Use only recommended buffers since 
DECP is instable in presence of  for exsample TRIS. 
The electrophoresis unit and the SEMI-DRY BLOTTER II 
should be treated for RNAse comtamination by soaking in 
DECP treated and autoclaved water. 
	Acrylic is NOT resistant to DECP.

5x MOPS buffer
	morpholinopropane-sulfonic acid	41.9g
	sodium acetate			6.8g
	Na2EDTA				1.9g
	ad 1000ML

10x loading buffer
	Ficoll 400			5.0g
	Na2EDTA		10ul 1M solution
	bromophenol blue		40mg
	xylene cyanole FF		40mg

1.	Prepare gel as follows:
		agarose		2.0g
		Water			104ML
	melt in microwave or by boiling, cool to 50!C
	add	37% formaldehyde	30ML	
		5xMOPS buffer	34ML
	pour the gel.
2.	Prepare the sample by mixing
		RNA (<20ug)		4.5ul
		5xMOPS buffer	2.0ul
		37% formaldehyde	3.5ul
		formamide		10.0ul	
	incubate the mixture at 55!C for 15 min.
	add 2 ul 10x loading buffer
3.	Apply the sample and run the electrophoresis for 
	approximal 3 hours at 200V (10x15x1 cm gel).
4.	Incubate the gel in 0.1xMOPS for 30 min.
5.	Assemble the transfer sandwich as in Step 15 (DNA procedure) 	but with the
filter papers soaked in 0.1xMOPS.
6.	Assemble the SEMI-DRY BLOTTER II and electrotransfer
	 at constant current for 30 min.
	(use approximal 3-5 mA/sq cm of gel).
7.	Wash the membrane in 2xSSC for 2 x 5 min.
8.	Process the membrane according to the manufactors instruction.



References
Fleming JE & Paull TT, Biotechniques 6,924-929 (1988)
Thomas PS PNAS 77,55201(1980)
Southern E Meth. Enzymol 68,152-176 (1979)
Maniatis T, Fritsch EF & Sambrook J Molecular Cloning Cold Spring Harbor 1982

*************************************************************
SEMI-DRY transfer of proteins
============================

The methods are based upon the tricine-SDS-Page system  
(gger & von Jagow 1987) due to its excelent separation 
down to MW of 1000. A homogen acrylamide concentration 
matrix is used (16% T, 3% C).
Different transfer buffers can be used. Originally  a 
e-caproic acid buffer was used in the paper of Kyhse-Andersen,
 but we have had better general transfer with CAPS buffer.
General trasfer conditions are 120 min at 0.2mA/cm2 in 
10 mM CAPS pH 11 20% methanol.

'CAPS transfer buffer
CASP (3-(cyclohexylamino)-1-propanesulfonic acid		4.42 g
ad 1600 ml  water adjust pH to 10.0 with 2 N NaOH
ad methnol								400 ml
water ad 2000 ml

Not all proteins/peptides  may transfer sufficiently.
Alternative buffer systems can be used try:
1.	Lower pH
	25 mM N-ethylmorpholine pH 8.3 or
	50 mM borate pH 8.0
2.	Reduce methanol concentration to 5%
3.	Ad 0.001-0.005% SDS to the transfer buffer
4.	Increase the current

References.
gger h & von Jagow G (1987) Anal Biochem 166,368-379
gger h, Aquila H & von Jagow G. (1988) Anal Biochem 173,201-205
Matsudaira P (1987) JBC 262,10035-10038
Kyhse-Andersen J J Biochem Biophys Meth 10,203-209
Barkholt V & Jensen AL (1989) Anal Biochem 



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