HELP !!!! mysterious cloning problem

Sergio sergioal at bbm1.ucm.es
Mon Oct 22 04:04:42 EST 2001


Just a (maybe silly) hint: are the expected restriction fragments different
size?. Are the enzymes compatible?. What's the size of the two bands observed in
the SmaI restriction and the single band observed in the BglII restriction?.

TINA ha escrito:

> 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 !
> Tina




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