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cloning problem


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3 replies to this topic

#1 deespike

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Posted 17 May 2009 - 07:40 AM

hi

im trying to digest a vector and insert with ecori and hindiii enzyme and perform ligation.The Tm value for the EcoRI and HindIII cut sites is 16C .I have tried ligatin in lower temperature range but didnot suceed .Can I use the temperature between 16C and 25C to balance with the efficiency of ligase or it is not required ?Im confused with the temperature that is optimal for successful cloning.

Plz can anyone help??

#2 HomeBrew

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Posted 17 May 2009 - 06:06 PM

Though doing the ligation at a temperature selected to maximize your chances of successful ligation helps, it is nowhere near the most important thing. Several things vastly outweigh the temperature at which you do your ligations in predicting a successful outcome -- the quality of your DNA (both vector and insert), the quality of your ligase and ATP in the buffer, and the competency of your cells, for example.

I do all my ligations at 16C, I've never even calculated the Tm of a restriction site...

I guess what I'm saying is that if your cloning experiment failed, it is unlikely that the failure was due to your using a 16C ligation temperature.

#3 T C

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Posted 18 May 2009 - 03:00 PM

Hi,

I agree with Homebrew, there could be other factors...Whats the size of fragments?

TC

Chk this too

http://www.protocol-...amp;#entry24508

#4 swanny

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Posted 18 May 2009 - 05:57 PM

Have you tested the components to make sure the DNA is correctly digested? Try religating both the insert and some DNA ladder (in separate tubes, of course!!). The insert should make at least 3 bands (monomer, dimer and trimer), higher is better; the DNA ladder should just move up. If it doesn't, the ligase is off. If the insert doesn't go to at least a trimer, one enzyme isn't completely cut.

Treat the vector with phosphatase to prevent recircularisation.
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