Cloning Fails-- Trouble shoot please

Sorry for the earlier post with such a serious error. I know EcoRI does
not create a blunt end.
This is of coursenot a blunt end cloning. Anyway I am not able to get a
bit success in this.
I have repeated three times this cloning and every time I get numerous
colonies none of which contains any insert. Actually I am trying to
clone a EcoRI cut insert into a vector that is almost 9kb. I check my
clones by colony pcr from the overnight grown single colonies from the
bacterial plates.
This time I set up a control while performing transformation.
I transformed
-vector EcoRI cut and purified, I got three colonies
-vector EcoRI cut, column purified and CIP treated and column purified,

I got 7 colonies
-vector EcoRI cut, column purified and CIP treated and column purified
plus insert EcoRI and purified in the ratio of 1:4 and 1: 6. I got
more than 100 colonies.


Can anyone explain what went wrong? None of the colonies contained the
insert when I checked them using primers specific for the insert.
However, control pcr worked. Control contained the insert, EcoRI
purified fragement

On 8 Nov 2005 03:21:15 -0800, "vijaykr" <[Only registered users see links. ]> wrote:


Have you actually done a miniprep, digest and see if it releases a
fragment? Or sequencing? It may be that your colony PCR failed for
reasons other than not having an insert.

Did you ligated the EcoRI cut vector (your first control)? I find it
hard to believe that you only get 3 colonies if you have ligated it.

On 8 Nov 2005 03:21:15 -0800, "vijaykr" <[Only registered users see links. ]> wrote:


Your colony counts suggest that it might be working. Two questions:

(1) Is your insert free from contaminating vector? You coudl test by
transforming the bugs with just the insert.

(2) . Perhaps your PCR is just not working from DNA isolated from the
bugs. Try miniprepping some of the colonies and checking them with an
EcoRI digest "the old-fashioned" way.

Nick

--
Nick Theodorakis
[Only registered users see links. ]
contact form:
[Only registered users see links. ]

vijaykr wrote:



You need to be more specific:
1. size of vector
2. size of insert
3. ligation conditions (%PEG)
4. how many colonies you screened
5. how you quantify vector and insert
6. mass quantity of vector and insert

BTW...CIP is not a magic solution as it tends to chew up overhangs if
you are not careful...

Cheers

Austin

Yesterday, I did the miniprep and EcoRI digestion. Its pretty clear
that the plasmids are not having insert. There was a single band that
corresponded to the size of the vector.
As a control to check EcoRI activity I transformed the EcoRI cut vector
after column purifying without ligating and another control to check
CIP activity by religating CIP treated vector and transformed this one
also. The former control gave three colonies and the later one showed 7
colonies.

Minprep followed by EcoRI digestion did not release the insert. Infact
it was just recircularised vector that I got in 15 colonies I screened
so far per plate.

On 9 Nov 2005 06:37:19 -0800,
vijaykr ([Only registered users see links. ]) wrote:

You haven't answered these questions that were posed earlier by someone
else:

1) How large is the insert? The vector is 9 kb, correct?

2) How many nanograms of each are you using in your ligations?

3) You're ligating PCR products, correct? How close are the EcoRI sites
to the 5' end of each primer?

4) Did you confirm that the PCR product was cut with EcoRI at both
ends? To do so, self-ligate the cut PCR product at 37oC for a couple
hours, and run it out on a gel. You should see a ladder. If the
majority of the ligated products are trimers and higher, both ends
have been cut. If the majority is a monomer, then EcoRI has cut
neither end of your PCR product. If the major product is a dimer,
then only one end of the PCR product has been cut. The experiment
I've just described makes the assumption that the 5' ends of your
PCR primers are not phosphorylated (which is likely to be the case,
unless you've specially ordered phosphorylated primers).

AC
--
Aawara Chowdhury, Shelter 5300, NOLA.

On 9 Nov 2005 06:37:19 -0800, "vijaykr" <[Only registered users see links. ]> wrote:


I would suggest that you sequence the plasmid you get from the
miniprep. From your description alone, the ligation must have
worked, although it is difficult to tell here from afar if you have
done anything wrong that might have caused the problem (it is always
easier to see someone actually doing it to identify any problem).

The insert may have deleted itself by recombination (so sequencing is
useful for checking), so check and compare the sequence of both your
insert and the plasmid to see if there is any possibility of that. A
very unlikely possibility is the presence of intein in your insert
which can somehow cause the problem. Someone I know had a problem
with an intein which he didn't know was present (he solved the problem
by identifying and removing the intein sequence), but I don't know if
intein can cause the deletion your insert.

If your insert is large and the plasmid is high copy number, a useful
way of doing screening for insert is to pick the colonies from plate,
lyse the cells and run the cell lysate in the gel. You then look for
size difference between the plasmids with insert and the one without
(run one without insert). In your case this method might be useful
and you can screen a large number relatively easily and quickly.
There are many protocols around for this screening method, have a look
in Google, if you can't find them, let me know.

Try directional cloning and see if it makes any difference.