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Cloning problems - 16Kb and 1.7Kb cloning problems (Jul/14/2009 )

Hello , Im having problems with a cloning experiment and lm getting to the suicidal point. heheheheh.

I have a 16kb vector which is linearized with SacII (cohesive end) and my insert is 1.7kb long, with SacII cohesive ends. Eventhough this could sound like an easy ligation is not working. I have tried everything.

Ive tried everything vector:insert ratio 1:1, 1:3, 1:5, insert:vector ratio 1:1, 1:3, 1:5. and I cant get positive clones.
First, I linearized my vector and tried to purify it with the Qiagen qiaquick colum system... doesnt work for Dna fragment that long, gives a poor yield, so im performing now electroelution, the yield is better. After the digestion, and purification, I perform a desphosphorilation with CIAP, 1hr 37C, then quantify the DNA and then the ligation with my 1.7Kb insert

Ive been using T4 DNA ligase from Fermentas that provides a 10X buffer and allows to use more DNA in a less total amount of reaction.

Id like to know if someone knows something about this, or long fragment ligation problems?
or if someone knows something about an special bacterial strain that can handle this experiment? or a Ligase that is better suitable for this?!
I would really appreciate the info
Greets

-soldierhxc-

So, do you get colonies? Are they religation of the parent vector? How are you transforming the ligation product? How much DNA do you add to your ligation mix, in what volume?

-phage434-

Can you tell us what controls you carried out at each step and the results?
Attached File

-jiajia1987-

It sounds like your plasmid is religating, that happened to me-if you are not getting bands after a colony PCR then the plasmid must be religating. After you cut your plasmid and run it in a gel do a dephosphorylation, then clean that up-should be a protocol in the gel extraction kits, and then redo the ligation.

-PCR Novice-

phge434: Generally this is the protocol that Ive been following:


Digest vector (16Kb) SacII --> Purify vector by electroelution (since qiagen columns sux for dnas larger than 10Kb) --> Resuspend in 20uL h20 --> CIAP --> Inactivation of CIAP 15min 65C --> Quantify

Digest insert (1.7Kb) SacII --> Purify insert with qiagen colum --> Quantify

Ligation: Generally I use final volume of 10uL with 200ng total (vector and insert) at different ratios. 50:150 100:100 150:50 etc, I have used many different ratios, i also tried the recomendations by promega (http://www.promega.com/biomath/default.htm) 100:10, 100:30 and 100:50 but nothing seems to work.

The couldnt be religation of the parent vector because I obtain a 3kb plasmid does not correspond to the 16kb nor the 17.7kb that im looking for. My insert does not have any AB resistant

Im transforming XL-Gold through termal shock 30min ice --> 45 secs 42C --> 5min ice --> add SOC --> 1hr 37C 300rpm --> plate on SOB with AB


jiajia1987 : Controls and results:

Digest vector and transform --> no colonies
Digest vector - CIAP - and transform --> no colonies
Digest vector - CIAP - Re ligation and transform--> no colonies
Digest (liberate) insert from plasmid and transform --> no colonies
Ligation insert and plasmid and transformation --->colonies but with a plasmid ok 3Kb

-soldierhxc-

Hi There,
I know this sounds like a really simple thing, but in our lab, it gets really hot during the summer months, and the temperature was preventing the fragments from ligating. Make sure your room is at a decent temperature range, because as soon as we cooled it down, the ligations worked properly!

Jenn

-jennlou2-