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help on difficult cloning

help on difficult cloning - Protocols and Methods Forum

help on difficult cloning - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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Old 05-27-2004, 03:48 PM
Chunxin Wang
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Default help on difficult cloning



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

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