ligation/cloning problems

Wolfgang Schechinger wolfsc at ibms.sinica.edu.tw
Mon Mar 25 09:36:53 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.

Dear Jens,

Assuming that both RE sites are present, the enzymes are ok and there is no
other theoretical knot in your construction, it should work.

I think using the kit for every step is overkill, you're probably loosing too
much DNA with all the purifications.
You may do everything together in one setup. NEB Buffer 2 with a little BSA
works fine (I am right now doing that to check some ligations). Also add .5 to
1ul of CIP or better SAP to the vector while digesting. .5ul (approx 10U) of
each enzyme will work fine within 1 or 2 hrs (you may incubate longer though).

Then run a gel and cut out your insert and also check an aliquot of the host
for complete cutting. The vector digest you can use without gel purif., since
both enzymes cut in the polylinker (do a phenol/chloroform extraction or use
e.g. the Qiagen gel kit to remove the enzymes).

For the ligation use different ratios (say .5 ul of vector (eluted with 50ul)
and .5, 2, 8 ul insert (also eluted with 50ul, in my experience it's difficult
to get accurate concentration of this DNA by UV). add ligase buffer, heat for
30 sec to say 50 degC, cool and add ligase (.5 to 1 ul in a 1 ul setup). Don't
forget ATP if not included in ligase buffer or add some additional ATP if
you're not sure that the ATP in the buffer is ok.

Do not heat inactivate the ligase. Make sure that your bacs are competent (use
.1 ug of original plasmid to make sure you get a tight lawn of colonies)

 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,

Actually, I wouldn't expect problems. Xho and Xba still cleave nice (and work
fine in my hands) when there is just 1 base left except the recognition site
(according to NEB catalogue tech/textbook section). Maybe digest overnight to
be sure since one enzyme probably needs to fall off to give room for the other.

Good luck,
Wo
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Dr. Wolfgang Schechinger
Institute of Biomedical Sciences
Academia Sinica, Taipei, Taiwan

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