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Problems with ligation


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#1 Lfs

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Posted 09 December 2015 - 07:36 AM

Hello!

 

I'm new in the fiel of cloning and I am struggling in the ligation thing...ohmy.png

 

I would like to know what is the best molar ratio for ligating a 302bp insert into a 2021bp vetor. I read that when you have a small insert, you should use a huge amount of it in order to get a good result. In the other hand, some people say, that you should't, because you could form dimers between inserts. So I'm very confused. I applied the basic formula and did some online calculations, but I got different results everytime.

 

Is there another way to do it? What is the logic behind insert/plasmid lenght, ligation efficiency, etc ...

 

Also, I have 40 ng of 302bp insert in 20ul (total) and 400ng/ul of the vector. If the final volume of my ligation reaction is 10 ul, should I increase this volume based on my DNA (if I use all the 20ul) or decrease it in order to stay between 10 - 20ul of ligation reaction? The volumes of enzyme, buffer and BSA should be proportional to the final volume?

 

Usually, for a 10ul mix (T4 DNA ligase Promega):

 

0,1 - 1ul enzyme (should I use 1ul?)

0,2 BSA

2 ul Buffer

insert

vector

water

 

Thank you very much!

rolleyes.gif

 

 



#2 labtastic

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Posted 10 December 2015 - 08:14 AM

A lot of people make up rules for how to do molecular biology that are largely based off of anecdotal evidence derived from their unique system.

 

Take most of these "rules" as rough starting point guidelines when you don't have anything else to go by. This goes for the "you need X : X molar ratio for optimal ligation efficiency" rule. You need to empirically determine what works best for your particular system. 

 

In other words, try 3 ligation reactions keeping fixed plasmid and adding different amounts of insert. For a 10 ul reaction I might do:

 

H2O (to make 10ul total volume)

1 ul digested, gel-purified vector

1, 2 and 4 ul of digested, gel-purified insert

2 ul 5x ligase buffer (I have never used BSA for a ligation)

0.5ul of T4 Ligase

 

2 hrs to overnight at room temp. 

 

Transform 2-4ul of ligation reaction into 50ul of competent cells.

 

Plate all cells after recovery. 

 

One question though: are you sure you have 400ng/ul vector? Did you digest and gel-purify this vector? This is an oddly high concentration for digested plasmid after gel purification. Your 2ng/ul concentration for digested, gel-purified insert seems oddly low as well.


Edited by labtastic, 10 December 2015 - 08:15 AM.


#3 Andrea Fortina

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Posted 20 June 2016 - 07:24 PM

For most standard cloning (where the insert is smaller than the vector) a 3 insert : 1 vector ratio will work just fine. But for your reaction, your insert concentration is much lower than the vector. So you could try only use 0.1-0.2ul vector and use the rest of the volume for the insert. Use 10* buffer, do not add water.


Andrea Fortina

Genius only means hard-working for all one's life.

CreativeBiomart, Recombinant protein expert

 

 





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