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CLONING PROBLEM!!!


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#1 biochemistry23

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Posted 18 May 2010 - 03:11 AM

hello everyone!!!!!

I have a problem with my cloning. I cut my vector with BamHI, I do heat inactivation, I purify it with a Nucleospin column and then I run a gel, where I have enough DNA. After that I make the ligation and next day I run a small amount of the ligation and I see no vector at all!!!!

Any ideas??????? Thank you!!!!!

#2 phage434

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Posted 18 May 2010 - 04:00 AM

How much DNA are you loading on your gel. Two things are different about your post-ligation gel band -- it likely has much less DNA (you've diluted it and run only a fraction), and likely your ligation has produced complex products which are spread out over the gel, making individual bands difficult to see. 10-20 ng per band is typically easy to see on a gel. Less than that is tricky. Running a post-ligation gel is not the easiest way to tell if a ligation is working (except for very controlled circumstances).

And you can't heat inactivate BamHI.

#3 biochemistry23

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Posted 18 May 2010 - 10:24 AM

How much DNA are you loading on your gel. Two things are different about your post-ligation gel band -- it likely has much less DNA (you've diluted it and run only a fraction), and likely your ligation has produced complex products which are spread out over the gel, making individual bands difficult to see. 10-20 ng per band is typically easy to see on a gel. Less than that is tricky. Running a post-ligation gel is not the easiest way to tell if a ligation is working (except for very controlled circumstances).

And you can't heat inactivate BamHI.


Actually that was the only ay to check if there is any DNA in my ligation reaction, because I get no colonies after the electroporation and I have checked every step one by one. So, I run 4ul from a 10ul ligation reaction.

#4 fishdoc

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Posted 18 May 2010 - 11:30 AM

How much DNA are you loading on your gel. Two things are different about your post-ligation gel band -- it likely has much less DNA (you've diluted it and run only a fraction), and likely your ligation has produced complex products which are spread out over the gel, making individual bands difficult to see. 10-20 ng per band is typically easy to see on a gel. Less than that is tricky. Running a post-ligation gel is not the easiest way to tell if a ligation is working (except for very controlled circumstances).

And you can't heat inactivate BamHI.


Actually that was the only ay to check if there is any DNA in my ligation reaction, because I get no colonies after the electroporation and I have checked every step one by one. So, I run 4ul from a 10ul ligation reaction.




Use your ligation reaction as template for PCR. Use primers specific to the vector just outside the cloning site. A small product indicates empty vector, a larger product indicates an insertion, no product points to no ligation.

What concentrations of DNA are you adding to your ligation reactions?

#5 biochemistry23

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Posted 18 May 2010 - 01:10 PM

How much DNA are you loading on your gel. Two things are different about your post-ligation gel band -- it likely has much less DNA (you've diluted it and run only a fraction), and likely your ligation has produced complex products which are spread out over the gel, making individual bands difficult to see. 10-20 ng per band is typically easy to see on a gel. Less than that is tricky. Running a post-ligation gel is not the easiest way to tell if a ligation is working (except for very controlled circumstances).

And you can't heat inactivate BamHI.


Actually that was the only ay to check if there is any DNA in my ligation reaction, because I get no colonies after the electroporation and I have checked every step one by one. So, I run 4ul from a 10ul ligation reaction.




Use your ligation reaction as template for PCR. Use primers specific to the vector just outside the cloning site. A small product indicates empty vector, a larger product indicates an insertion, no product points to no ligation.

What concentrations of DNA are you adding to your ligation reactions?


I usually add 100ng of the vector.

#6 fishdoc

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Posted 18 May 2010 - 03:08 PM

I usually add 100ng of the vector.




You're not inserting anything, just trying to ligate the empty vector?

#7 biochemistry23

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Posted 18 May 2010 - 11:28 PM

I usually add 100ng of the vector.




You're not inserting anything, just trying to ligate the empty vector?


Exactly! I am just trying to religate the vector in order to check me new ligase.




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