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Ligation Mistake

ligation

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#1 SeekingResearcher

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Posted 03 April 2015 - 12:22 PM

I performed a ligation earlier that will be used in a transformation tomorrow. However, I made an error in reading the protocol and mistakenly added twice as much 10X T4 Ligation Buffer in mixing with my plasmid, diluted duplex oligo, dH20, and T4 ligase. Specifically, the protocol asked for 50ng plasmid, 1ul oligo, 1ul ligase, 1ul buffer, and dH20 for a total of 10ul system.

 

However, instead of 1ul I added 2ul of 10X T4 ligation buffer. I understand that the main purpose of the buffer is to provide ATP, but I'm unsure if twice as much would actually result in any noticeable negative effects.

 

Unfortunately, I have no more plasmid to be able to redo the ligation. I still intend to perform the transformation tomorrow, but would like to know maybe what I can expect or if it's even that good idea to continue. I'm probably overreacting, but this particular experiment is pretty important.



#2 GNANA

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Posted 03 April 2015 - 12:59 PM

I would go ahead with the transformation too.  its not a big mistake to make your ligation fail completely.

 

good luck.


I always had an alternate hypothesis....


#3 bob1

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Posted 03 April 2015 - 01:21 PM

if you have 2x buffer in the ligation mix it most likely will fail, as the salt concentrations are usually critical for enzyme function. This is the reason you need to purify the DNA before trying the ligation. ATP provision is another thing. Too much there probably won't hurt, though in theory it could provide enough energy to allow ligation of non-complementary ends.



#4 GNANA

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Posted 03 April 2015 - 02:59 PM

I agree with bob1 that too much salt concentration would fail the ligation reaction, but i think, the 2x has around 120mM of Tris-HCL and Mgcl2 combined. Moreover, NEB claims that ligation will work in any of the 4 RE or PNK buffers, when supplemented with ATP, which do have higher salt than ligation buffer.  so i still believe, if other steps had gone well i would expect positive colonies in the transformation plate.


I always had an alternate hypothesis....


#5 SeekingResearcher

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Posted 03 April 2015 - 05:21 PM

I appreciate the replies bob1 and GNANA. What both of you said definitely makes sense. These definitely aren't the ideal conditions for the ligation, but I'm feeling a little more optimistic about there still being a few positive colonies. I'll continue with the transformation tomorrow and hope for the best. Thanks!


Edited by SeekingResearcher, 03 April 2015 - 05:22 PM.






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