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fill-in remaining nicks in recombinant plasmid

fill-in remaining nicks in recombinant plasmid - Protocols and Methods Forum

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  #1  
Old 04-25-2005, 05:00 PM
Vagelis
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Default fill-in remaining nicks in recombinant plasmid



Hello everyone, I would appreciate some help on this one:
I am working with a 5kb plasmid from which I remove a 114bp oligo and
replace it with a modified 114bp oligo. The vector is dephosphorylated
prior to ligation with the phosphorylated insert and thus my
recombinant vector contains two remaining nicks.
Now, when I transform E.coli with my recombinant vector the two nicks
are repaired inside the bacteria, as is evident from the sequences I
get back from the colonies, which suggest that the modified oligo is
successfully incorporated.
However, when I transfect Ad293 human kidney cells with my vector, the
cells somehow (exonucleases?) chew my oligo, as the sequence data
suggest.
Do you know why this happens and how I can avoid it?
Is there any way I can seal the remaining nicks before transfection
into the human cells?
How about Taq polymerase, would it seal the nicks?
Any suggestions are welcome,
Thank you in advance,
Vagelis, Leicester
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  #2  
Old 04-25-2005, 06:30 PM
Austin P. So (Hae Jin)
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Default fill-in remaining nicks in recombinant plasmid

Vagelis wrote:



Hmmm...is there a reason why you don't just do a prep from your
transformed E. coli and transfect with that?

Anyway...I think a solution would depend on the length and type of your
overhangs that you are using to insert your oligo...

cheers

Austin
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  #3  
Old 04-26-2005, 12:51 PM
Vagelis
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Default fill-in remaining nicks in recombinant plasmid

I cannot use the plasmid from the transformed E.coli because a DNA
adduct is incorporated into the 114mer and the recombinant vector is
used for adduct site-specific studies.
The vector is digested with the XhoI and BamHI restriction enzymes
which both produce sticky ends:

XhoI
5'....C^TCGA G....3'
3'....G AGCT^C....5'

BamHI
5'....G^GATC C....3'
3'....C CTAG^G....5'


"Austin P. So (Hae Jin)" <[Only registered users see links. ]> wrote in message news:<d4jd1p$i8a$[Only registered users see links. ].ca>...
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