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Protocols: ES Cell Culture - Gene Targeting - Protocols

Genomic DNA:
Restriction Enzyme Digests, Agarose Gel Electrophoresis,
and Southern Transfer (Blotting)

NOTES:	Until the Southern transfer is complete, 
use only those materials reserved for Genomic use.  
These include: Eppendorf tubes, all buffers, enzymes, 
and a fresh dH2O supply.

	When working with genomic DNA, always snip off the ends of the 
pipette tips to prevent the DNA from being sheared and keep the average 
size in excess of 80-100 kilobases (this is only necessary until the 
digest is complete).

	ALWAYS wear gloves when working with ethidium bromide (EtBr)!!  
It is an extremely potent carcinogen.

Restriction Enzyme Digest

	1.	Rotate DNA samples 5-10 rpm for 30 minutes.

	2.	Measure the concentration (ug/ul) of each sample using the 
fluorometer.  Calculate the volume of DNA equivalent to 5 ug of DNA based 
on the concentration (volume to use in ul = 5 ug divided by the concentration).


			Sample	Conc. (ug/ul)	Equiv. of 5 ug DNA (ul)*

			A134		0.770			6
			A135		0.935			5
			A136		2.140			2

			*Round to the nearest whole number

	3.	Set up a series of Eppendorf tubes for the digest and add the 
volume of each DNA sample (ul) equivalent to 5 ug of DNA (see step 2 above).  
Bring the volume of each tube to 20 ul with dH2O.

	4.	To each tube add 20 ul of the enzyme mixture composed of:

	--	5U enzyme/ug DNA (i.e., 25U enzyme/tube);

	--	Buffer appropriate for the enzyme being used, to a concentration 
of 2X (since this will give the buffer a final concentration of 1X when added 
1:1 to the 20 ul DNA);
	--	dH2O to bring the volume to 20 ul.

		An alternative to preparing an enzyme mix for each sample 
tube is to prepare a stock enzyme mixture that can be aliquotted into 
the sample tubes.  To prepare this, simply calculate the total volume 
required for all sample tubes [(# tubes + 1) x 20 ul] (the extra tube 
is for good measure).  Then calculate the fractions of this volume needed 
for enzyme, buffer, and dH2O.


	20 samples are to be digested.  Therefore, (21 x 20 ul) = 420 ul 
total are needed for the stock enzyme mixture.

	If a HindIII digest is being performed, 25U enzyme/tube is required.  
For the stock solution, (21 x 25U/tube) = 525U enzyme total are needed.  
The commercial HindIII comes in a stock concentration of 20U/ul.  
Therefore, [525U / (20U/ul)] = 26.25 ul, rounded to 26 ul HindIII are 

	HindIII uses Medium Buffer (50 mM NaCl).  The stock buffer is 
10X in concentration; 2X is desired.  Therefore, (2 / 10 ) = 0.2 or 
one-fifth of the total volume needs to be contributed by buffer 
(0.2 x 420 ul = 84 ul M Buffer).

	The remaining volume (420 ul - 26 ul HindIII - 84 ul M Buffer) = 310 ul 
is composed of dH2O.

	After mixing the ingredients of the stock enzyme mixture, aliquot 
20 ul into each of the sample tubes, bringing the volume of each tube to 
40 ul total.

	5.	Incubate digests overnight @ 37o C.

Agarose Gel Electrophoresis

	1.	Prepare the agarose gel and the electrophoretic buffer.  Each 
large gel will require 2 L of buffer.

	a.	Make 1X TBE (generally from a 5X TBE stock solution) and add 1 ul 
of Ethidium Bromide (EtBr--10 mg/ml) per 100 ml of buffer (i.e., add 10 ul 
EtBr per liter of buffer).  Stir on a magnetic stirrer to insure a uniform 
distribution of the EtBr.

	b.	Take 1/5 of the total volume of buffer (400 ml for each gel) 
and add 0.7% (w/v) electrophoresis grade agarose. Microwave until the 
agarose is completely dissolved and is clear.  Cool at room temp. 
for approx. 10-20 minutes.

	c.	Securely tape the ends of the gel tray (large) and insert 
the comb at one end.  When the agarose has cooled slightly, pour the 
gel to a depth of approx. 3/8" to 1/2".  Allow the agarose to solidify, 
about 30 minutes at room temp. (this can be accomplished faster by 
putting the gel tray @ 4o C when pouring the agarose).  Remove the comb 
after the gel has completely solidified.

	2.	While the gel is solidifying, remove the digest(s) from 
the incubator and add 7 ul of genomic loading buffer to each sample.  
Spin for a few seconds to insure that the loading buffer has mixed 
with the sample.

	3.	Fully submerge the solidified agarose gel in electrophoretic 
buffer with the wells closest to the negative (black) electrode.  
Load the full volume of each sample (47 ul, or the maximum volume 
allowed by the wells) into a well.  Also load two wells, one on 
each side of the samples, with 20 ul of BstE II u marker.

	4.	Secure the gel box lid and connect the electrodes 
(red to red, black to black) to the large power source.  Run the 
gel @ a maximum of 60-65V until the desired fragment size separation 
is obtained.  (Small bubbles should be visible in the buffer, 
indicating a closed circuit.)

Southern Transfer (Blot)

	1.	Once the gel has run long enough for the desired 
fragment separation to be obtained, photograph the gel.

	2.	Trim the gel to 1 cm of required area of transfer and 
measure its dimensions (l x w).

	3.	Soak the gel twice @ room temp. in 0.25 M HCl, 5 minutes 
per soak.  Change the HCl solution between soaks.  Do not soak more 
than 10 minutes total.  This partially hydrolyzes the DNA by acid 
depurination and aids in the transfer of large DNA fragments.

	4.	Denature the DNA by soaking the gel in 0.4 N NaOH for 
20 minutes.  Proceed with step 5 while the gel is soaking.

	5.	Cut one piece of GeneScreen Plus (transfer membrane) and 
two pieces of Whatman 3MM paper to fit the dimensions of the gel 
(measured in step 2).  GeneScreen Plus has a natural curl when dry.  
Using a ball point pen, mark the concave side of the membrane with 
an *.  This is the side to which the transferred DNA will be bound.

	6.	Carefully wet the membrane with dH2O by capillary action.  
Let the membrane soak for 15 minutes.

	7.	Set up the transfer apparatus as shown in Figure 1.

	a.	Put a sponge in a glass dish and soak in 0.4 N NaOH.  
Squeeze the air out of the sponge.  The level of the NaOH should be 
below the top surface of the sponge.

	b.	Cut 2 pieces of Whatman 3MM paper to the width of the 
sponge and 4-5" longer than the sponge.  Place these on top of the 
sponge with the ends trailing into the 0.4 N NaOH.  These serve as wicks.

	c.	Carefully invert the gel onto the transfer apparatus 
(with the open end of the wells facing down).

	d.	Cover the gel and dish with plastic wrap, insuring that 
no bubbles are trapped between the gel and plastic wrap.  If there are 
bubbles, use a 10 ml pipette to roll them out.

	e.	With a sharp scalpel blade, cut a window in the plastic wrap 
slightly smaller than the dimensions of the gel (about 0.5 cm from each 
edge of the gel).

	8.	Take the transfer membrane from the dH2O, briefly submerge 
in 0.4 N NaOH, and apply so that the marked (*) side is against the gel.  
The membrane must be kept wet at all times.  Roll out any bubbles trapped 
underneath the membrane.

	9.	One at a time, take the 2 pieces of Whatman 3MM paper 
cut in step 5, wet in dH2O, and place on top of the filter (membrane), 
rolling out any trapped bubbles.

	10.	Apply a stack of dry paper towels on top of the transfer.

	11.	Allow to transfer overnight.

	12.	Take down the transfer and rinse the filter in 0.2 M Tris 
pH 7.5, 2X SSC.  The blue dye-front should appear on the filter.
13.	Air dry the filter @ room temp. between 2 sheets of Whatman 
3MM paper.  The membrane is now ready for hybridization.