5 replies to this topic

[soldierhxc]

Hello , I´m having problems with a cloning experiment and l´m 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.

I´ve 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 37°C, then quantify the DNA and then the ligation with my 1.7Kb insert

I´ve 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.

I´d 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

[phage434]

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?

[jiajia1987]

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

[PCR Novice]

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.

[soldierhxc]

phge434: Generally this is the protocol that I´ve been following:


Digest vector (16Kb) SacII --> Purify vector by electroelution (since qiagen columns sux for dna´s larger than 10Kb) --> Resuspend in 20uL h20 --> CIAP --> Inactivation of CIAP 15min 65°C --> 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.c...ath/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

I´m transforming XL-Gold through termal shock 30min ice --> 45 secs 42°C --> 5min ice --> add SOC --> 1hr 37°C 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

[jennlou2]

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