ligation/cloning problems

jens
Mon Mar 25 08:52:11 EST 2002


Hi,
I have some problems with cloning a 4kb cDNA(MDR1) insert into a 5.4 kb
pcDNA3.1(+) Expression-vector. I use the pgem3zf(-)-XbaI-MDR1.1 to obtain
the cDNA. I found out that the first 100bp of the cDNA are not necessary for
translation and the hold a XhoI restriction site. So I cut the cDNA with
XhoI first, purify DNA with Qiagen gel Extraction Kit and then cut the DNA
with XbaI. After that I treat the cut vector with CIAP (even though this
should not be necessary), do gel  and again extract the DNA with Gel
Extraction kit.
The next step is a ligation carried otu at 16°C overnight with a maximum DNA
concentration of 10ng/µl. Last step is a transformation. The result is
always the same. Most of the time I get no colonies and if I get some the
E.COli only hold pcDNA3.1(+) without any insert. When I looked up the MCS of
pcDNA I saw that there are only 2 bp between the restriction sites of XhoI
and XbaI, is that a problem?
Thanks in advance
  Jens


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