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Thermo scientific T4 DNA ligase product help

ligation T4 dna ligase

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#1 KhaleesiDany

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Posted 06 February 2015 - 12:54 AM

Greetings

 

I have been struggling to ligate a 555 bp insert into a 6.1kb vector and everything is failing i get no colonies or i get one which when screened gives msomething else..i tried to just religate the vector and i still get no colonies even when i do not dephosphorylate the vector...I  even tried with 500 ng vector and a 1:5 vector insert ratio. nothing! i checked the t4 dna ligase bottle from thermo scientific and it says it is 5 Weiss U/µL 1000 Weiss U (200 000 CEU).. the protocol says for a sticky end ligation i need to add 1 weiss U for a 20 ul reaction... all my life i have been using 1 ul for every ligation i have done, so if i add 1 ul does that mean im adding 5 weiss units instead? how do i add 1 weiss unit instead 

 

 

thanks in advance



#2 phage434

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Posted 06 February 2015 - 08:32 AM

As I noted earlier, your problem is almost certainly not the ligase. Continue to use 1 ul, and check the ligase buffer.



#3 Meg P. Anula

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Posted 11 February 2015 - 10:23 PM

I used NEB T4 DNA ligase instead. I suppose it's fine as long as you put more ligase than it should be. I used 1 ul as well.  

 

What temperature you use to incubate and for how long? The safer and most optimum is usually 16c overnight. 

 

Also referring to Sambrook's Molecular Cloning, I prefer to "relax" the dna (vector and insert) in 45c for 5 mins and then chill on ice before i put in buffer and then ligase....

 

And have you prepare a control? To make sure the competent cells are working.



#4 KhaleesiDany

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Posted 14 February 2015 - 12:24 AM

I used NEB T4 DNA ligase instead. I suppose it's fine as long as you put more ligase than it should be. I used 1 ul as well.  

 

What temperature you use to incubate and for how long? The safer and most optimum is usually 16c overnight. 

 

Also referring to Sambrook's Molecular Cloning, I prefer to "relax" the dna (vector and insert) in 45c for 5 mins and then chill on ice before i put in buffer and then ligase....

 

And have you prepare a control? To make sure the competent cells are working.

yes competent cells working fine, ive tried NEB but i get like 2-5 colonies which is better than non so i will screen hopefully its something







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