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ligation problem


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#1 deespike

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Posted 16 June 2010 - 08:55 AM

Hi all

I am having some problem regarding to ligation - sticky and blunt ligation.

I am doing both the ligations for different insert and vector . The stick end ligation is for a vector of 4.6kb and 1 kb insert . i perform with neb ligase i use 2 units/ul of enzyme in 16C overnite in rapidligation buffer(promega) that contains 5% peg8000. The vector and insert preparation is also from kit. But i donot get any result of ligation in whatso ever I:V concentration i take. There comes simply no colony after ligation (my comp cells are okay no problem with transformation).

Similarly , my another ligation is blunt ligation between a vector of 12kb and insert of 3kb. I use the same enzyme and buffer at 22c but no result.

When i am using kit prepared insert , vector ,and rapid ligation buffer why is there no ligation, alos enzyme is fresh.
can anyone please tell me the reason . I have been doing this for several months with no result.

Any suggestions is appreciable.

#2 Clare

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Posted 17 June 2010 - 05:06 AM

You haven't accidently phoshorylated your vector AND your insert have you? If this happened you would not get any colonies.

Clare

Hi all

I am having some problem regarding to ligation - sticky and blunt ligation.

I am doing both the ligations for different insert and vector . The stick end ligation is for a vector of 4.6kb and 1 kb insert . i perform with neb ligase i use 2 units/ul of enzyme in 16C overnite in rapidligation buffer(promega) that contains 5% peg8000. The vector and insert preparation is also from kit. But i donot get any result of ligation in whatso ever I:V concentration i take. There comes simply no colony after ligation (my comp cells are okay no problem with transformation).

Similarly , my another ligation is blunt ligation between a vector of 12kb and insert of 3kb. I use the same enzyme and buffer at 22c but no result.

When i am using kit prepared insert , vector ,and rapid ligation buffer why is there no ligation, alos enzyme is fresh.
can anyone please tell me the reason . I have been doing this for several months with no result.

Any suggestions is appreciable.



#3 deespike

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Posted 17 June 2010 - 07:47 AM

i havent phosphorylated any of them. But i am unable to follow your suggestion
please be clear.

Thanks

#4 ranvi

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Posted 17 June 2010 - 10:31 AM

You phosphorylate the Vector to prevent self closure. You do it only for the vector. ie treating with CIP or Sap (phosphatase), you cant do the same for the insert then there wont be any colonies for ligation

#5 molstudent

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Posted 17 June 2010 - 12:03 PM

dont you guys mean de-phosphorylate?

#6 ranvi

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Posted 17 June 2010 - 12:49 PM

yeah removing the phosphate group we normally say as cip treatment or Sap treatment

#7 Clare

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Posted 18 June 2010 - 03:57 AM

dont you guys mean de-phosphorylate?


Ooops! yes we do - I always get my P's mixed up ^_^

Clare




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