Problems cloning adaptor
kang at msvax.mssm.edu
kang at msvax.mssm.edu
Fri Sep 15 23:35:42 EST 1995
In article <bjanssen-1209951033460001 at hall-pmac8100.tamu.edu>, bjanssen at bio.tamu.edu (Bart Janssen) writes:
>I am having problems trying to clone an adaptor into vector, I'm still
>doing some controls to check all the steps but if anybody has some advice
>on adaptor cloning I'd certainly appreciate it.
>This is what I'm doing. I have a 7kbp plsamid with a unique SalI site
>that I don't want to destroy, what I want to do is add a KpnI site next to
>the the SalI site. I designed an oligo adapter with the following
>I annealed the two oligos (~1ug of each) by heating to 90 C for 10 min and
>then allowing to cool slowly to room temp (by just turning off the heat
>block and going to lunch). I then kinased the annealed adaptor (T4 PNK,
>Boehringer) and purified using Bio101's Mermaid kit.
>My vector was digested overnight with SalI and then dephosphorylated using
>CIP and purified using wizard DNA clean up. I have cloned several other
>SalI insert fragments into this vector using SalI cut-CIPed vector with no
>problems so I think the site is OK, however the SalI site does lie between
>the RK2 oriT and oriV in a binary vector which might cause problems.
>Ligations (overnight at 16 C, BRL Ligase and buffer plus added rATP) were
>set up with ~50 ng of vector and from 1 ng to 100 ng of adaptor and an
>additional ligation set up with ~10 ng of vector and 300 ng of adaptor as
>well as a vector only control.
>On my transformation plates I see a couple of colonies on all plates.
>I still need to check both the kinase and the ligase, but I doubt that
>they are the problem since the rest of the cloning I am doing is working.
>My next step (I think) is instead of cloning an adaptor in, I can design
>PCR primers to amplify a fragment from the SalI site around to a PstI or a
>SacII site and introduce the KpnI site using the primers. This would
>allow me to purify cut vector and let me see the insert fragment on a gel
>before the ligation.
>If anybody has any good ideas as to what else I could do I'd be very
>grateful (sorry no prizes, I'm stil paying PhD debts too:)).
I developed very simple method
Please read Biotechniques 15, (1993,Oct),659
"One-step insertion of oligonucleotide linkers and adapters to DNA
using unphosphorylated oligonucleotide"
So far I changed more than 100 enzyme sites using this methods.
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