Consistent subcloning from agarose (finally !)

the End jgraham at bronze.ucs.indiana.edu
Wed Apr 7 15:04:15 EST 1993


In response to Paul's queries:

1) TAE only (not TBE)
2) isopropanol bath in a -70, or just set in the -70
3) thaw at 20-37 C doesn't matter as long as it doesn't remelt.
4) spin at room temp

The whole thing is pretty forgiving as can be seen. I would hesitate to involve
ANY steps involving binding the DNA to resins or even trasfering tubes, as 
this is source of sample loss. Glassmilk worked for me a few times, but not
EVERY time with all size fragments.

As far as visualizing the bands, be SURE you have a 360 nm peak transillum.
Ours is an old UVProducts model. Next to it is a new mid-range (302 nm peak)
model, also from UVP, that is very commonly seen in labs. This one gives
sterile transformant plates every time. It also give much brighter bands
on analytical gels. I have not tried methylene blue, but I would not want
to allow the diffusion that might occur in staining/destaining especially
with short fragments. For this reason I only run these a short way into the
gel before slicing out the bands. The sensitivity of EthBr I need as 
errors in quantitation in everyday estimations are common, as well as 
partials and when partial digestion is desirable. Methylene blue might
be helpful in those big batches, or when a long wave bulb is not available.
I believe you can use the $10 bulbs (Fishcer) in your medium wave
transilumnator.

Jim
J. Graham



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