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Ligation left for weekend at room temp

cloning ligation T4

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4 replies to this topic

#1 YuriyG

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Posted 17 March 2014 - 02:38 AM

I was rushing around and I accidentally left my ligation which was intended as an overnight for the weekend on my bench. It included one blund end ligation and one circularisation of a linearised vector. I'm wondering whether anyone who has done this before has had any luck with the products, or should I start again. As I don't want to then transform and waste time and bacteria.



#2 jerryshelly1

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Posted 17 March 2014 - 06:53 AM

We have had people who have left vial of T4 ligase out overnight and the enzyme was still active. I would think the ATP in the buffer would be the greatest factor. After two days at room temperature, the ATP is probably degraded. With that said, I do not think there will be any problem with your ligation. If you left out the enzyme and the buffer, you should do some controls to see if they are still active.

 

Also, I don't know how you usually do a transformation but is there anyway you can run your ligated product out on a gel and visualize the expected band? Maybe do a gel extraction? 



#3 perneseblue

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Posted 17 March 2014 - 02:45 PM

Should be fine. I would transform it.


May your PCR products be long, your protocols short and your boss on holiday

#4 phage434

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Posted 17 March 2014 - 08:51 PM

I agree it is worth a try, but the issue is the possible presence of DNAse contamination. The only reason DNA is not as much of a problem as RNA is the ease of inhibiting and removing DNAse with EDTA or heat. A reaction such as a ligation will have plenty of Mg ions to allow DNAse activity to happen, so if it there, it will be active.



#5 YuriyG

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Posted 18 March 2014 - 07:23 AM

Thanks, I'm going to give it a go as we only have a little bit of the vector left anyway and I'd be wasting it if I chucked the stuff. A few vials of DH5α aren't too dear.







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