blunt end !!!
Ian A. York
iayork at panix.com
Mon Dec 23 16:41:39 EST 1996
In article <3GFJP2$lLb at bbs.life.nthu.edu.tw>,
O·R·ÓÃè¤lªºmutant! <isa.bbs at bbs.life.nthu.edu.tw> wrote:
>I have tried blunt end ligation for two mouths.
>But the cloning was still not done.
>Does anybody have good method?
I've had pretty good success with blunt cloning for the past while (of
course, having said that, it will never work again). Here are some things
to keep in mind.
(1) As with any cloning, make sure your vector and insert are clean. Be
careful with the purification. I use Geneclean routinely; there are many
other ways of purification that work as well.
(2) Make sure your blunt ends really are blunt. I suspect this is more
myth than reality, but it won't hurt to polish your blunt ends with T4 DNA
Pol or Klenow.
(3) Dephosphorylate your vector. This makes a big difference in cutting
down religation. I use calf intestinal phosphatase (CIP) from New England
Biolabs and clean it up with Geneclean after the dephosphorylation. Be
*very* sure that you follow the instructions for the CIP; unlike many
other enzymes in cloning, the concentration of this enzyme vs. DNA
concentration is critical for good results. Calculation the number of
moles of DNA ends and use the appropriate amount of CIP. You'll probably
find that you need to dilute the CIP 10- or even 100-fold to get the right
amount. Too much will mess up the ends.
(4) Use a fairly wide range of insert:vector ratios. I run out small
samples of both on a minigel and eyeball the relative concentrations, then
try to get insert:vector ratios of 1:1, 5:1, 10:1, 20:1 in the ligation.
Usually the best ratio is around 5:1 or 10:1, and the other ratios on
either end help make sure that even if your estimate is off you can hit
those. Some people use much higher insert:vector ratios, like 50:1; I
don't think that's necessary or even very useful.
(5) You may be able to do some more tricks to reduce the number of
self-ligated vectors. If, for example, you've cut your vector with EcoRV
and there are no EcoRV sites in your insert, then you can digest your
ligation (after the appropriate period) with EcoRV; this will only cut
vector with no insert (because the insert will disrupt the EcoRV site, of
course), and the linear vector will be very inefficiently transformed into
bacteria; this enriches for those vectors with insert.
I don't worry about playing with ligase concentrations (I use New England
BioLabs T4 ligase), ATP concentrations, adding PEG, ligation temperature,
or anything like that; the above approach has allowed me to get blunt ends
in first try for the past couple of years.
Good luck; hope this helps.
Posted and mailed.
Ian York (iayork at panix.com) <http://www.panix.com/~iayork/>
"-but as he was a York, I am rather inclined to suppose him a
very respectable Man." -Jane Austen, The History of England
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