a03m at biologie.uni-bremen.de
Fri Dec 13 11:20:17 EST 1996
"Chang,jihun" <j6544 at unitel.co.kr> wrote:
>Thank you for your reply.
>Your kind advices moved me.
>Thank you very much for your reply.
>But I made big mistake.
>Oh! my GOD!!!
>I'm trying to ligate not 'Hind III'-digested genomic DNA ,but 'HaeIII'.
>I'm very sorry for my mistake.
>Please, show me your kindness once more.
>Condition of my competent cell(DH5a) is not so bad.
>In 82mm plate, 200-300 blue colonies & 15-20 white colonies.
>I did not treat CIP into vector.
>I digested genomic DNA with HaeIII overnight, and second boosting 4-5hrs
as you have lots of blue clones, you should dephosphorylate your
vector for the next run. There where many postings in the past,
indicating that Sma1 sometimes makes trouble. Take EcoR5 instead and
do not cut over night!!
Last not least, I personally prefer to cut and dephosphorylate without
changing buffer (CIP works fine in 'high' restriction buffers), and to
gel purify the vector. So no uncut or 'hypercoiled' vector
contaminates your ligation reactions!
P.S. Also check the following hints that where posted recently. No
more could be said about ligation conditions!
In article <57mb54$7sk at bignews.shef.ac.uk>, Kevin Mulcahy
<K.Mulcahy at sheffield.ac.uk> writes:
> Does anyone know if the presence of TE buffer in a ligation reaction can
> affect the reaction causing ligation failure? If so, how much TE (in a
> 20µl reaction) would cause inhibition, or is more appropriate to
> dissolve the DNA fragments in water in order to avoid the presence of TE
> in the ligation reaction? Lastly, if water is recommended, should I
> adjust the pH of the water to 7.5-8.0 (since our lab water is pH 6-6.5)
> so that the DNA will dissolve more easily?
> Many thanks for your help,
> Kevin Mulcahy.
I routinely dissolve my DNA in TE for ligations (and sometimes even
0.5x TBE) and use 7ul + 2ul 5x buffer + 1ul ligase. My ligations
generally work fine. If I recall correctly, the final concentration
of Mg in the ligation is 10mM, so the 1mM EDTA in the TE shouldn't
have any effect. There are many reasons that ligations fail, and I
can't even pretend to have figured them out. Here are a few of the
things that I DON'T do anymore.
1) Don't digest more than 3hr (Do use the appropriate amount of
enzyme) (SAP of vector in the cut is also very desirable - according
to a recent posting on this newsgroup, it works only in hi salt
buffers like BM buffer B or H)
2) Don't ligate without gel purifying
3) Don't use Qiaex, Qiaquick, or any other kit for gel extraction
(Do just elute slice thru a spin filter - works great).
4) Don't worry about ratios (enough is enough - it either will work or
5) Don't use 16 C, Do ligation at RT for 1-3hr or in the fridge O/N.
(I use GIBCO ligase and Buffer, which has PEG in it).
Hope this helps
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