HELP !!!! mysterious cloning problem

TINA anfortin at
Fri Oct 19 18:51:26 EST 2001

Hi everybody,

I'm trying to introduce a HA Tag to a gene already present in a pSVL vector.
I introduced this Tag via PCR with Pwo DNA pol. After this I added "A"
nucleotide with TAQ DNA pol. in order to sub-clone in the TA cloning vector
pCR 2.1.This PCR product was purified by QIAEX method and ligated into the
pCR 2.1 vector. After transformation of INV cells (supplied in the pCR2.1
kit) I've got an acceptable number of colonies and I screened 24 of them by
PRC to know which of these clones have the insert (gene + Tag, 304 bp).
Next steps : -liquid culture O/N
                    - miniprep (Qiagen with columns)
                    - analytical digestion to know which clone have the
insert in the correct orientation
It is the second time that I do this entire procedure.
The first time was not good because there was a mutation revealed by the
sequence analysis. This mutation originate from an error in one primer used
in the initial PCR. Other mutations were also detected, probably du to the
use of TAQ DNA pol. to generate the Tag.
The second time I started with Pwo, but the problem is at the analytical
digestion step!!! I have to cut with  Sma I and Bgl II, Sma I is unique in
the insert and Bgl II is unique in pCR 2.1 vector. Surprise ... no digestion
of clones, I have got only one band on agarose gel instead of two !!! Sma I
control gives two bands ... its not normal  and Bgl II control gives one
band , its OK.
What's happen ??? The PCR reaction tells me that the insert is present but I
cannot see it by digestion !!!
Nevertheless I have got good digestion results at my first trial !!
I tried two things in order to find a solution but nothing:
- I have started new culture (and new miniprep) from colonies choosen first
and from newly selected colonies
  After by PCR the original colonies seems to have loss there insert and the
newly selected ones all !!!! seems to have the insert.
- I tried to digest with Eco RI to see the insert in an other way than PCR
(there is an Eco RI site at both side of the multiple cloning site of pCR2.1
vector). Again I've got only one band instead of two as indicator of insert.
So the Sma I enzyme is not deteriored.

Please help me !
It is my first experience in cloning and also I'm a beginner in Mol. Biol.
This situation is not good to help an Immunologist like me to like Mol.
Biol.  !!!  :))  :))

Thanks in advances to take of your time to read this long message !

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