help on difficult cloning

Hi,

I am struggling with the cloning of several protein sequences and
started to look up in internet if someone came across of similar
problem. And apparently your problems were similar to mine (below I
copied the message I found in
[Only registered users see links. ]). So I was
wondering if you could resolve your problem with cloning at that time if
yes - how?
My problem is somewhat similar to yours in the sense that after ligation
reaction I get all or almost all clones positive by PCR but later on
after plasmid purification non of my clones seems to have insert.
If you could help me with this matter I would really appreciate it.

Regards,

Olga Senkovich
University of Alabama at Birmingham

Thu May 27 10:48:27 EST 2004

Hi all,

It might be long as I tried to describe my problem in detail. Hope you
can help me out.

I've been trying to make 9 constructs with yeast 2-hybrid vectors
pGADT7 and pGBKT7. For cloning of 7 constructs I had no problem but the
last 2 constructs took me more than one month without success.

My strategy for all the constructs is to PCR amplify the genes of
interest with cloning sites introduced into the primers. For R1 gene I
used NdeI and XhoI to clone into the AD vector and for R2a I used EcoRI
and BamHI to clone into the BD vector.

With AD-R1 ligation products, I was able to get very limited
transformants ( less than 20, even less than the AD self-ligation
control). However, colony screening with PCR showed that all of them
were positive. But miniprep of all of them produced no insert at all.
Later on, I changed one of the cloning site XhoI with ClaI and easily
got the correct clones with right insert.

For BD-R2a ligation products, I was able to get lots of transformants
(10 times more than the BD self-ligation control). PCR screening showed
that only 1 out of 25 are positive. Miniprep of the positive clones
showed that all the positive clones have the inserts of right size.
However, sequencing of those clones only revealed that 15-20 n.t were
missing from the 3' junction, even the 3' primer sequence was
truncated. I sequenced three of them and they all have the similar
problem. One of the clones even has the orientation changed. I examined
the 3' primer sequence I used to amplify the gene by PCR and found it
has a high homolog to the sequence right before the ATG of the gene.
So, I redesigned the 3' primer sequence and redid the cloning process
again. This time, with the new BD-R2a ligation products, I only got
less than 10 transformants (less than the BD self-ligation control).
However, PCR screening of all the colonies showed that they are all
positive. But digestion of miniprep of all the clones produced no
insert or much larger than expected inserts (4 or 5 times bigger).

What should I do?

Black

On May 4, 12:38 pm, "Senkovich, Olga" <[Only registered users see links. ].edu> wrote:

Hi Olga,
I assume all your PCR controls are working (especially NTC) . In that
case you might be dealing with a plasmid that is unstable (toxic) .
Few thinks might help - grow at RT or bellow. When you pick colonies
to grow try to see if there are different phenotypes - small vs. large
(probably large ones are the ones that spit the plasmid). Finally you
migh try different bacteria - I know Stratagene sells few strains that
claim to be better for unstableproducts.
Hope that helps,
Peter