help on difficult cloning

[peter]

On May 4, 12:38 pm, "Senkovich, Olga" <senkov... from cbse.uab.edu> wrote:
> 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 inhttp://www.bio.net/bionet/mm/methods/2004-May/098289.html). 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

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