Darcey --
So far you have received much good advice. You do not explain why you
are trying to blunt clone a PCR product, and if you do not have a
particular reason to blunt clone (eg extreme poverty, a particular vector
you must clone into), it does make more sense to clone your PCR product
into a commercially available T-overhang vector (or you can make such a
vector yourself) and then you can cut out large quantities of your PCR
product from a miniprep and clone the PCR product into the vector you are
interested in.
However, assuming that you really do want to blunt clone your PCR
product:
As mentioned in this newsgroup, you must:
phosphorylate your primers or your PCR product
Clean the A's off your PCR product (eg with T4 polymerase)
Dephosphorylate your vector.
In terms of size restriction, it is invariably much easier to clone tiny
inserts (eg 200 bp) than big inserts (>3 kb). PCR products are
notoriously difficult to clone. When cloning blunt, it can help to use
more ligase (eg 1 u/20 ul reaction). The key to most ligations is the
cleanliness of insert and vector. If you are gel purifying your insert,
you must be certain that no inhibitors from the agarose are present in
your final prep -- not easy to verify.
In terms of vector quality, there are some controls you can run. If
you are certain that your vector is good, you are halfway there:
1) when your vector is digested, it should be digested fully. If a
percentage of the vector remains uncut (and you cut out the cut portion)
you may still notice a significant decrease in ligation efficiency. Gel
purification is not magic, and it does not get rid of everything "but the
band". If your digestion is incomplete, either your enzyme is finicky
(not true of most standard enzymes) or vector miniprep is dirty.
YOu can test your vector with the following controls:
Make up the following ligation reactions, all in your usual ligation
buffers, and transform them all after the ligations are complete:
1) Your dephosphorylated vector, use no ligase.
2) YOur dephosphorylated vector, with ligase
3) First kinase your vector, then purify it (Phenol:CHloro -> Ethanol),
then make a ligation reaction with ligase
4) YOur dephosphorylated vector + your PCR product (with phosphorylated
ends, ragged ends polished with Klenow or T4, gel purified if necessary) +
ligase
5) Any normal plasmid you have lying around (eg pUC or bluescript)
In an ideal world, you will get no tranformants in tubes 1 & 2, tons of
transformants in tube 3, and a fair number of transformants (your final
product) in tube 4. If tube 1 has transformants (ie more than 3), your
vector is not cut or you have not purified it completely from uncut
vector. If 1 has no tranformants but tube 2 has quite a few, it means
that your dephosphorylation reaction is not efficient enough. Of all the
enzymes I have worked with, phosphatase can be the most temperamental and
go off most unexpectedly. The onl solution for this problem is to buy or
borrow a different batch of phosphatase. If tube 3 does not have
transformants, if may mean that your vector is no good, that it has
contaminants that prevent it from ligating, or it could mean that your
kinase step did not work (less likely). If tube 5 produces few or no
transformants, then your tranformation is failing (rather than your
ligations). Unless you have used a plasmid which does not have the
correct resistance gene (eg ampicillin), this implies that your bacteria
are not competent. The fastest solution to this problem is to buy
commercially available competent bacteria -- expensive, but necessary. If
all the tubes work as expected except tube 4, then your vector is fine,
and the problem is in your PCR product. The rest of the advice and
protocols you have received should guide you in polishing your PCR
product, kinasing it, and cleaning it (either by gel or just phenol/chloro
ethanol).
All the best,
Harry
Ed Smith wrote:
> I have been trying for several months now to blunt-end ligate a PCR
> product into a Bluescript plasmid and am having no luck at all. It
> worked once.... I am using Bluescript KS- (3kb) and a 3kb insert
> which has been treated with Klenow to make it blunt-ended. Does
> anyone have any advice or suggestions on how to make this work for me?
> Are there size restrictions on the fragments that would make this
> difficult, or certain ratios of insert to vector I should know about.
> I have tried 1:1 ratios, and various insert or vector excesses. Both
> have been gel isolated, agarase treated, and purified. I don't use a
> kit, but have been using T4 DNA ligase from Fisher, and my ligations
> are done at 14 degrees C. Does anyone have any "foolproof" protocols
> or suggestions? Thanks.
>> Darcey Smith
>darcey at juno.com
--
Harry J. Witchel, Ph.D.
Dept. Physiology
Medical School
Bristol BS8 1TD
England
Harry.Witchel at Bristol.ac.ukhttp://www.bris.ac.uk/Depts/Physiology/Staff/hw.htm
