David LePage's Gene Targeting Protocols

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Digesting Genomic DNA In 96-Well Plates

After resuspending overnight the DNA is ready to digest. If the wells were dense then there is enough DNA for two gels, so you can load half of each digest and still have a nice bright smear. Freeze the remainder of each digestion as a back-up.

-Add cocktail to your DNA giving yourself enough for 2-3 extra rows. Mix gently by stirring the DNA with the cocktail and drawing up once or twice (DNA is very viscous and this will give you a sense of how thick your samples are--useful for comparing after they've digested overnight).

-Seal each plate with plastic wrap and put the lids back on. Stack several plates and wrap them together as well. Put in a humidified container and place in a 37 degree warm room or oven overnight.

Cutting Cocktail


20 µL

10 x buffer

4 µL

BSA (100 mg/ml)

0.4 µL (100 µg/mL)

Spermidine (50 mM)

0.8 µL (1 mM final) {make concentrated spermidine in sterile ddH20, aliquot and store -20: no other preparation required}

RNase (10 mg/mL)

0.2 µL (50 µg/mL; this is standard RNAse ala Maniatis, i.e. heated to eliminate trace DNases)

Restriction enzyme

10-40 units (I prefer Boehringer[Roche] or New England Biolabs enzymes. Always buy new tubes of enzyme for this so there is no chance of contamination by sloppy lab-mates and because you'll use a lot of it anyway. If the enzyme is not a reliable type, such as EcoRI, HindIII, BamHI, XbaI, etc., it is definitely worth trying it out first on some spare DNA prepared by this method to be sure it will work reliably).

Sterile ddH2O

q.v. 40 µL total volume.

-The next day check a few wells by drawing them up with a P200 and seeing that they drip smoothly down. If they seem too viscous add some more enzyme (high concentration enzymes come in handy for this) up to 1/10 the total volume. Alternatively you can run a few microliters of some rows on a mini-gel to see that you get a nice smear.

-Assuming the digests are complete add 5 µL 10 x loading dye (we usually use glycerol dye) and load 25 µL of it into a 0.7% agarose/0.5 x TBE gel. We have very large 12 x 8 inch gels which easily accommodate three rows of our thin 36 well combs. By loading 32 samples on each row of the gel you have room for a whole plate plus markers.

-SPECIAL NOTE ABOUT BRB: This building has terrible environmental controls, and it never fails that a brisk wind is always blowing in the bay in which you are loading a bunch of Southerns. Be aware that because some of the samples will still be a little viscous (they'll still work, they're just slightly thick) you'll want to be careful about the wind, since if a trail of sample reaches the top of the buffer surface, the wind will suck the sample out of the well. To counter this I always have the safety lid for the gel box resting on the box, just slightly below the row of wells that I am currently loading. If a sample is gooey and has a long trailer of goo floating up, as quickly as possible I cover the gel box with it's safety lid and wait for the trailer to sink down below the surface (believe me this works and can save your sample). As a bonus having the lid just below the row you are loading will guide you where to load as you robotically proceed with endless samples.

-There are two ways to keep the samples from running into each other. Run fast at 2 V/cm for about an hour until the samples have entered nicely, then separate the gel into three slabs with a clear space between them. Turn the juice down to 1 V/cm and run overnight. Alternatively you can start them running late at night and get back to the lab early the next day to separate the gel into three slabs.

-The gels are run overnight at 1 V/cm. I have easily detected large bands in the 22kb range after an overnight run. My usual practice for detecting 3 kb or higher is to run until the first dye (bpb) runs out and the xylene cyanol dye is about halfway down each slab.

-After running I always try to take a picture of each slab, since this provides a valuable back-up if you should mis-label any of the blots: the occasional blank wells from false positives can then be used to orient the autorads with the original picture.

-We use the basic Schleicher and Schuell Nytran plus membrane with alkaline transfer, but I see no reason to switch to this if you prefer something else and are confident that it works well.


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