Is my DNA clean enough for a succesful ligation? - (Aug/08/2007 )

I'm having considerable problems with the ligation of sub-clonned constructs. Could the problem be the quality of my DNA?

I'm ligating inserts into +pcDNA and -pcDNA. After digestion, I gel extract the vectors using the Omega Bio-Tek E.Z.N.A Gel Extraction Kit and strictly following their protocol. The +pcDNA has a 260/280 ratio of 1.4 and a 260/230 ratio of .45. The -pcDNA has a 260/280 ratio of 1.4 and a 260/230 ratio of .12.

Thank you

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Have you checked your purified DNA fragments on a gel? I don't even bother using a spec to quantify fragments, as the readings tend to be inaccurate. Running a few uL of purified DNA on a gel will allow you to estimate the concentration and check the purity of the DNA. If you have nice clean bands the ligation should work.

A little more info may help people answer your question. Have you checked to make sure you're restriction digests are working ie: sequential vs. double digest, etc? Are you getting no colonies at all, or just empty vector?

Its usually unlikely that a kit is the problem. There are a number of other things that could effect quality and ligation effeciency.

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The ratios sound pretty bad, but more likely something else is wrong. I vote for UV exposure of your DNA. You should minimize the time your DNA is exposed to UV, and make sure you are using long wave (365 nm) UV. Better, use visible blue light. Also, it is common for people to try ligations with large amounts of DNA. Try doing a ligation where the amount of DNA in the ligation is in the 10-30 ng range, and keep the volume of added liquid carrying the DNA as low as possible (this dilutes contaminants). Are your transformation controls working?

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your 260/230 ratios are pretty awful for a decent preparation for subcloning. 2 probabilities : salts in your prep or presence of solvents. You're not efficiently dry them before resuspending in ethanol.
So i would suggest, do 2 washes and check that pelletis properly detached from the tube. (in case of the possibility salts are also contaminating)
Wait longer time for etoh drying.
respsuend.

note : ethanol decrease the efficiency of ligation !

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Thanks for the reply.

To fred:
The kit uses a silica binding column, not etoh precipitation. So maybe I should put the kit aside and try etoh precipitation? or maybe use the kit, but then phenol chloroform extract the DNA?

To phage and smitity:
In troubleshooting my ligation problem I thought I would address one issue at a time- thinking it wouldn't be much help to ask, "hey guys, why is my ligation not working". But since you guys asked here goes:

After gel extraction, I have analytically checked my vector and insert and they look good. Before I was getting mostly empty vector (although one of my constructs really did work once). So I took the +/- pcDNA that I wanted that had very large inserts already cloned into them and double digested those. That way, I could make sure what I was cutting out was only empty, digested vector (the undigested construct was 3 Kb bigger). When I did this I started getting no colonies- but my postive transformation controls still worked. I don't see how I can prove that my insert is fully digested though- it is from PCR.

Phage, you're probably right. I think the vector that was cut away from the construct was exposed to too much UV (someone new to the lab did it- bad mistake on my part). So I redigested it and religated and am waiting for colonies as we speak. I ligated 100 ng- so I think you're right to go down to 30.

But if this doesn't work I'm really out of options.

thanks again. I'll let you know.

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As log as the ends of vector and inserts are not damaged by UV or over digestion and you have confirmed DNA on gel. The ligation should work.

Good Luck !!!

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Thanks. What if the UV damaged the AMPr gene of the vector? Then you might not get colonies, even though your ligation worked, ie. the backbone of the stickends was repaired.

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well you damage randomly the vector. And as the only pressure for selection is antibiotic selection, this is not a reference for estimating the damage done to the plasmid...

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hello people
Can you tell me the concept behind UV damaging DNA ends and that's affecting the ligation reaction. Also, tell me how long can we expose the gel to UV for observation of DNA? This is really new to me.. I've not heard of these kinds of troubles. Do feed me the details. Thank you

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Thymine-Thymine dimers

Nasty thing that happens when DNA is exposed to UV light.

The exposure time of DNA for ligation purposes to UV should be measured in seconds. I keep total exposure time to about 5 seconds, no more then 10secs. Long expose time will damage the DNA so badly that the vector won't replicated wiithin the cell, or worst yet replicate but the insert is riddled with mistakes.

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