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How to store a ligation reaction over the weekend?

Ligation Vector Plasmid DNA

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#1 joesep

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Posted 23 January 2014 - 02:43 AM

I wonder which is the best way to store my ligation reaction over the weekend.

I will do an overnight ligation at 15°C and I need the ligated vector on monday morning for electroporation of e. coli.

 

I tend to keep the ligation reaction at 4°C from friday until monday, then purify the DNA with a PCR purification kit, elute it in water and use it for the electroporation.

 

Would you rather freeze the ligation reaction on friday? Or do the DNA purification on friday and freeze it eluted in water?

 

I think freezing and thawing the DNA does damage it more than storing it at 4°C in the ligation reaction. 

What do you think?



#2 phage434

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Posted 23 January 2014 - 07:22 AM

(1) Why are you purifying your DNA. If you need to eliminate salt, then you can do this with drop dialysis, which is much easier and faster, and loses less sample.

(2) In my experience, ligations happen quickly (even with non- "quick ligase"). 5-15 minutes is all it takes, and can be done on the bench at room temperature.

(3) to answer your question, if I really wanted to keep a ligation over a weekend, I would do it by adding EDTA to chelate the magnesium, then store at +4, but why would you do that.



#3 joesep

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Posted 23 January 2014 - 09:47 AM

Thank you for your fast reply!

 

(1) Why are you purifying your DNA. If you need to eliminate salt, then you can do this with drop dialysis, which is much easier and faster, and loses less sample.

 

I actually never got to know another method than microcolumn purification. Is the loss of DNA really so much smaller when using drop dialysis? And is drop dialysis feasible with a quite large ligation reaction (approx. 150 µl)?

 

(2) In my experience, ligations happen quickly (even with non- "quick ligase"). 5-15 minutes is all it takes, and can be done on the bench at room temperature.

 

For standard ligations, I also use quick ligase for 5 min or normal T4 ligase for 30 min.

I only do the overnight ligation for library-construction, where I use a lot of DNA (approx. 1.6 µg for vector+insert), because I was told that this is more reliable then...

 

(3) to answer your question, if I really wanted to keep a ligation over a weekend, I would do it by adding EDTA to chelate the magnesium, then store at +4, but why would you do that.

 

So to inhibit DNAse activity, I would add EDTA to which concentration?



#4 phage434

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Posted 23 January 2014 - 02:12 PM

Add sufficient EDTA to counteract the Mg++ ion concentration.  Something like 10 mM should do for most reaction conditions, but you might want 20 mM just to be safe.







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