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Ligation failure? I ran some on a gel


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6 replies to this topic

#1 seanspotatobusiness

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Posted 03 May 2014 - 08:51 PM

I got no colonies after transforming some ligations.

 

I ran some of the ligations on a gel to see what they looked like. I've never done that before, so I'm not really sure what a successful ligation looks like.

 

The ladder is NEB 1 kb and the final lane is undigested vector. The lanes with bright spots had oligos and those without didn't (a control; didn't want colonies from that sample in any case!). It all looks like it's still linearised to me.

 

QHDE7dL.png



#2 bob1

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Posted 04 May 2014 - 01:31 AM

Its almost never worthwhile running a ligation on a gel - if you have checked all the components, then there shouldn't be too much of a problem (unless the ligase or buffer is off), the amounts you should be using for your ligation will often preclude being able to visualize it on a gel properly.

 

Having said that, what size is the insert and how big is the unligated vector?  If you ran some unligated on the gel, that would help diagnose the issue. Undigested vector isn't much help as this should often be mostly supercoiled and will run at odd sizes, though it may well contain some linearized or circular vector too.



#3 seanspotatobusiness

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Posted 04 May 2014 - 03:18 AM

The vector is 9.2 kb and the insert is about 30 bp. The quantity of vector was ~70 ng per ligation. I think the insert was too far in excess but the ligation ran two days at 15 C (16 C is optimal but we have a 15 C fridge for eggs). To me it all looks linear (it's the same as my linearised plasmid shown in the image below). I'm not sure why the third lane band is higher (maybe a well deformity?).

 

I'm using NEB T4 ligase. According to NEB I can carry out the ligation in 10 minutes at room temperature, or overnight at 16 C. I don't think the give any indication of the comparative efficiency of ligations for different durations or temperatures. I'd rather like it to be done in 10 minutes.

 

4qqjCGH.png



#4 phage434

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Posted 04 May 2014 - 07:05 AM

You will never see a 30 bp band shift on a 9.2kbp plasmid on a gel. Can you explain in more detail how you are planning to ligate this? You'll need a pair of complementary oligos, annealed, and 5' phosphorylated if you are trying to ligate them. You'll need a very dilute version of this annealed oligo, since you need roughly equimolar amounts, and the sizes differ dramatically. Ligations, if they are going to work, will likely work in 5 minutes, so two days is just a waste. Finally, almost all "ligation" problems (assuming the DNA is good) are really competent cell problems.



#5 seanspotatobusiness

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Posted 05 May 2014 - 06:22 AM

I was hoping to see a shift due to the circulation of the DNA rather than the 30 kb addition. The oligos are not phosphorylated but the vector should be so that can't be it, right? This time I diluted the oligos 125 times more and I'm hoping that will be the solution. I didn't realise that they sequester the ligase. It's also very useful to know that a ligation can be trusted to occur so quickly (thanks!) although at the moment I'm juggling so much stuff that I'm still inclined to the tubes at 16 C until it's convenient.

 

In case anyone else is interested, most of the way down this page appears to be a good explanation of the effects of temperature and time on ligation and why it might be done at a lower temperature and how that the necessitates longer incubation (I had mistakenly assumed that the enzyme was unstable/degraded quickly at room temperature): http://www.protocol-...osts/37153.html



#6 seanspotatobusiness

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Posted 06 May 2014 - 10:24 AM

Could the prolonged incubation at 16 C possibly be detrimental? My transformations yielded no colonies again. I've been using aliquots of buffer which have been stored at -20 C. Perhaps I should discard them and prepare fresh aliquots from the buffer which was supplied with my recent ligase purchase? Am I clutching at straws?



#7 bob1

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Posted 06 May 2014 - 12:56 PM

If you are annealing and then digesting the oligos, then they don't need to be phosphorylated; on the other hand, if you are generating the overlap by annealing, then they do need to be phosphorylated - otherwise they won't ligate.






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