UV and cloning, ETB (was: pcr prob)
Deitiker, Philip R
(by pdeitik from bcm.tmc.edu)
Fri Feb 29 11:31:01 EST 2008
I also try to have enough product so that the band can be light visualized and this is the benefit of optimizing PCR reactions.
The article in question I have a few problems with:
1. He used short wavelength
2. He used a bench transilluminator, at least for my transilluminator I have saturation of intense PCR bands on the low setting with very short time exposures. Essentially a 6 kb band of 2 ng saturates except at the lowest machine exposures.
3. In our lab we use a longwave length hand held illuminator and either from the side or far enough away from the band such that we can barely see it in an otherwise completely blackened room, I myself being incredibly nearsighted have my nose about 6 inches from the gel, so there is a major,
possibly ten fold difference in the level of exposure.
4. Most plasmids are arounds 4 kb or so, most inserts are less than 2 kb. A single mutation can destroy a plasmids viability and there are generally lots of sites in the plasmid that will do this.
5. I think it is foolish if people are having problems to divert them on the 'No UV' goosechase if they are having other problems. I have done experiments testing different cut cloned fragments to test efficiency from UV gels, meaning I had to individually cut 15 or 20 bands from the gel, single cut
plasmids always religated, even with some exposures over a minute. The efficiency did not appear to be related to the length of exposure time so much as the quality of band cut from the gel.
6. Cutting bands on a UV transilluminator is a good way of getting 'snow blindness' if the proper precautions are not taken, and I have seen this happen. Handheld UV sources are cheap and offer more flexibility (can be taken to a dark room or area, can be held at a great distance for intense bands)
and can be immediately diverted away once the cuts are made.
The article sounds more like one of these hypey advertisments than an actual science.
It is true even a momentary exposure to UV can result in a clone with a different sequence than desired, this was a frequent occurrence in the early days of cloning when sequencing was expensive. But there are all kinds of sources of sequence artifacts so that sequencing multiple clones needs to be
This thread has been going on for a very long time, really piling up the emails in my in-box. Is it possible to trim the volume of posts to relevant material, and catch multiple points in one post? I know there have been other complaints, the language has gotten out of hand, in my opinion. I voice
my opinion with others here, clean up your acts, people, my in-box is not a chat room.
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