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Help please! Directional cloning: colonies with WEIRD DNA?

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Help please! Directional cloning: colonies with WEIRD DNA?

Postby walkthewok » Sun Nov 30, 2008 5:41 am

Hello all,

I desperately need help with molecular cloning (directional, not blunt end). I've been going at this for 5 months, and I believe if I can overcome this hurdle I will be good to go. Please help, and thanks in advance!

Debriefing: Basically, I have a 16 KB vector containing an Arabidopsis (plant) gene sequence, that I am replacing with another Arabidopsis gene that I am interested in. The cut sites are Hind 3 and Pst I, and the insertion is directional cloning. I cut out a fragment and I insert a fragment ~300bp larger back into the vector.

Problem: After cloning, I am able to get colonies HOWEVER upon colonyPCR or regular PCR from miniprep DNA, THE LARGE MAJORITY OF COLONIES (maybe 65/70 colonies picked) that I get contain multiple bands, when they should only contain single bands. When I sequence the DNA of bacteria with these "double bands", I always get a "Failed Sequence". For the chromatograph, it appears that at the lowest setting, there's "multiple bands. "The primers used for these are primers that are reading off the vector, see the image for more details.

Question: WHAT is going on, and HOW can I avoid this?

Here is my protocol.

Vector Prep
1) Culture bacteria
2) Miniprep

Insert prep
1) PCR 6 tubes of 20 ul each per insert
2) Ethanol precipitate and combine PCR inserts of the same kind, concentrate into 20 ul

Then...
1) Digest ~1ug of vector, ~2ug of each specific insert with Hind 3 and Pst1 together, for 1-2 hours
2) Gel purify vectors and inserts (also, verify if vector was properly cut here because I can see
3) Ligate for 3 hrs at room temperature (this is according to protocol on ligation mixture) at 3:1 or 1:1 ratio
4) Transform via either electroporation or heat shock

I have transformed both via electroporation and heat shock, but both cases the majority of the colony PCRs yield these multiple band results.

Please help, and thanks in advance!

If you can't download the attachment, here's a link to the image:
http://img221.imageshack.us/my.php?imag ... cr2mi9.jpg
Attachments
colonypcr2.jpg
walkthewok
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Postby beautifulmind420 » Tue Dec 09, 2008 7:57 pm

Hi,

Just a question: Did you send your sample for sequencing to check if there is any mutation in gene of interest after transformation?
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