hansw at sci.kun.nl
Sun Aug 25 06:54:09 EST 2002
mlsulliv at facstaff.wisc.edu ("Michael L. Sullivan") wrote in message news:<firstname.lastname@example.org>...
> Make sure that when you visualize the fragments by UV, that they are
> not overexposed. A long-wave hand held UV lamp is what I use for
> visualizing fragments I am cutting out of gels-- this causes little
> if any damage to fragments. However, many UV light boxes can destroy
> fragments for cloning quite rapidly.
> >Hi, my friends,
> > I have sent this message once before.But till now I still cannot make it.
> > Please help me.
> > I'm in great trouble with my contruction of expression plasmids.The
> >vectors and fragments were all double digested by the same two
> >enzymes and target bands after electrophoresis were extracted out
> >from gel by the gel-extraction kit
> >from Roche or by freeze-thraw method.The extraction was very good
> >checked by selectrophoresis.For ligation,I tried many times and
> >many ways including rapid ligation kits from different companies and
> >individual reagents.I can't make it always.I am so discouraged.
> > Would anyone give me some suggestion and and information for my
> > Thanks in advance.
> > Huazhong
> > Wang Hua-zhong Ph.D
> > Cytogenetics Institute,
> > Department of Agronomy ,
> > Nanjing Agricultural University,
> > Nanjing,Jiangsu,P.R.China
> > 210095
I have encountered problems just like you and found some ways to
eliminate some problems.
1) Use CIAP on the digested vector: CIAP dephosphatises the 5' and 3'
ends of the digested vector resulting in lower self-ligation rates of
your vector and stimulates ligation of your insert.
2) Increase transformation efficiency by inactivating the ligation
reaction: incubate the ligation mixture for 20 minutes at 65 degrees
celcius (if you used T4 DNA ligase)
3) Instead of rapid ligation kits try to do this for example(it looks
very extreme given the concentrations but it worked in my case);
ligation reaction mixture:
1ul 10x T4 DNA ligase buffer
0,5ul 30 U/ul T4 DNA ligase (i know; 15 U is freaking much)
0,5ul 50 ng/ul vector
8 ul 150ng/ul insert
(TOTAL VOLUME: 10ul)
Incubate for 2hrs at room temperature and remember to inactivate the
ligation mixture by incubating for 20 minutes at 65 degrees celcius
4) transform 100ul of competent cells with 5 ul (inactivated) ligation
5) check your competent cells with a known concentration of vector. It
should yield about *10^8 CFU for each microgram of vector. (please
check this in the literature i do not know this for sure by heart).
Use 3 dilutions of 50ng/ul vector: 10^-4 , 10^-5, 10^-6
6) Be sure to have a high ratio of insert:vector
I hope you will be succesful. I have troubles with some genes even
when using the exact same protocol i used for succesful ligation (with
Goodluck and keep the faith!
REMEMBER: science is true, do not be misled by facts... ;-)
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