Blunt end ligation problem

Bert Gold bgold at ktb.net
Sat Dec 26 14:12:00 EST 1998


For a 5' overhang (on your insert), Klenow will usually work.  But for a 3'
overhang
it won't.  To do this successfully, you must know which you have...

If your insert contains a 3' overhang, try T4 Polymerase instead of Klenow for
the
'polishing'  as otherwise, your efforts to blunt the insert will have no
effect.

Next, spermidine does help the efficiency of the primary ligation to blunt
ends,
as does T4 RNA ligase in addition to T4 DNA ligase.  You can find references
to these ideas in many of the old Maniatis, Blue Bibles as well as the newer
Ausubel et al. manuals.

Third, do the primary ligation at high concentration with a high insert to
vector
molar ratio.  Guesstimate the amount of each by running a minigel
pre-ligation,
then use like 5:1 or 10:1 molar ratios of insert to vector in like 10 uL with
say
up to 250 ng of total DNA in a volume of 10 uL, for a primary ligation for
like one hour at 14 degrees C. (cold room).

Last, but not least, after you are done with your 10 uL primary ligation, at
least
one side of your insert should be stuck to the blunt end of your vector.
Check
this by running 1 uL of the 10 uL on a minigel next to your vector.  If the
ligation products aren't larger than your vector, you made some kinda error
somewhere.  (You should be able to calculate the exact size of your primary
ligation product in advance and figure out what kind of gel will resolve it
from
your vector alone).

At the end of all this, if you do, indeed have a primary ligation product, you
can
dilute the ligation mixture 10-fold (I usually add 86 uL of ligation buffer to
my
now, 9 uL primary ligation mix, then 4 uL of T4 DNA ligase then 1 uL T4 RNA
ligase) then go overnight at 14 degrees C.  The next morning, transform
and select.

You should get several colonies.

Bert Gold, Ph.D.
SmithKline Beecham

P.S. I give no guarantees.  This advice does not represent that of my
employing organization,
whose name is provided for identification purposes only.



Low Hai Sim wrote:

> Dear all,
>
>         I am currently facing a problem with ligating Klenow treated
> insert into a CIP-SmaI vector.  I have cloned two fragments into this
> vector before, both unidirectional sing two different enzymes.  Then I
> tried to clone in an EcoRI insert, so I Klenow treat, and CIP my vector.
> No transformants obtained.  I did the following trouble shooting :
> 1. Change ligase (2 different ligase)
> 2. Change competent cells (check competency with the vector itself)
> 3. Tried religation of vector.
>
>         When I tried religating the vector, I could only get a handful of
> transformants.  I know that this should not be the case, but I do not know
> where else could be the problem.  I changed the water, competent cells,
> ligation conditions etc.  I feel that if I cannot religate, something must
> be terribly wrong.  Am I missing something important here I-(  I refered
> to Sambrooks etc  Could someone please just send me a tried and tested
> protocol for blunt end ligation.  I will be very thankful.
>
> HS




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