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ligation--pls help!


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10 replies to this topic

#1 janani

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Posted 05 January 2011 - 02:36 AM

hi all,
after ligation of my vector and insert, when i run it on the gel, what exactly should it look like? should it run at the exact size that my clone is supposed to run? should it be a single band? can someone pls help me on this, since i am getting bands of high molecular weight, high above the ladder, near the wells. but transformation is not happening, i tried different inserts and vectors, did every possible thing like increasing/decreasing concentration, making new plasmid and insert from scratch a few times and restricting them all over, etc. my competent cells are perfect since my controls are perfect:( plss help.!

#2 phage434

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Posted 05 January 2011 - 05:18 AM

When you say that your controls are perfect, does this mean you have measured the competence of your cells? What is it? This is certainly the most common cause of failure. Are you using quick ligase or normal ligase buffer? Do you heat kill the ligase before running the gel? You may also have a construct which is lethal to the cells, so that even if the ligation is correct, the transformation does not work. We need a LOT more detail to begin to try guesses at the problem.

#3 GeneTurk

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Posted 05 January 2011 - 03:03 PM

I may recommend the following website for very important tips. They really saved a lot of time for my ligations, time, and reagents.

http://bitesizebio.c...ation-problems/

#4 janani

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Posted 06 January 2011 - 09:00 AM

I am trying to clone a 2kb microRNA promoter gene and its deletion constructs into a luciferase reporter. After ligation,i transform 4ul of ligation mix in 100ul of cells and get no colonies. I use NEB quick ligase and ligate for 10mins at 25C. If the ligation mix is run on the gel, i get a very high molecular weight band, for any insert and any vector. Is this the ligated plasmid's band? i never get it in the expected size. I always get it way above.

#5 janani

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Posted 06 January 2011 - 09:04 AM

i do not heat kill ligase before running on the gel.

#6 phage434

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Posted 06 January 2011 - 05:14 PM

Unless you are doing blunt ligations, I would switch to normal ligase buffer instead of the quick ligase. The PEG in quick ligase makes the gel give very high bands, and can't be heat killed. Normal ligase buffer can be heat killed and gives normal DNA bands, which can be used to analyze the ligation products. Usually, these are too complex to provide much insight, however, unless you are doing controls with carefully designed single-fragment self-ligations.

#7 genesquirt

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Posted 07 January 2011 - 12:20 AM

In my ligation experiments, I saw bands like aggregating to the size of the vector used (e.g pUC19/pUC18 etc.) It looks like they are forming a ladder in the picture. I think you are doing okay.

#8 janani

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Posted 08 January 2011 - 12:30 AM

Unless you are doing blunt ligations, I would switch to normal ligase buffer instead of the quick ligase. The PEG in quick ligase makes the gel give very high bands, and can't be heat killed. Normal ligase buffer can be heat killed and gives normal DNA bands, which can be used to analyze the ligation products. Usually, these are too complex to provide much insight, however, unless you are doing controls with carefully designed single-fragment self-ligations.


But have you faced this problem of very high bands? It gives the same sorta band for any ligation i do but ultimately transformation is not working. I do not get colonies. my positive and negative controls are giving the expected colonies. I do not know where else i am going wrong. pls help!

#9 janani

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Posted 21 January 2011 - 10:13 PM

pls help :(

#10 Rebio

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Posted 25 January 2011 - 11:11 AM

Electroporation with quick ligase do not work. PEG in quick ligase inhibit the electroporation. Try heat shock if you are using quick ligase.

Or switch over to regular T4 ligase and then use electroporation. You can also transform with Z.comp cells i.e. 5 minute transformation using regular T4 ligase.

#11 TheBalrog

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Posted 10 March 2011 - 02:12 PM

Hi Janani.
While trying to ligate into a 3.5 kb vector, I experienced 7-8kb bands when linearizing the DNA from minipreps, which corresponded to a 4-5 kb insert - not what I expected to clone :blink: Sequencing revealed that I had managed to clone a gene from a Pseudomonas bacterium which is commonly found in water! When I checked the sequence of the pseudomonas gene it contained an EcoRI and a NotI restriction site which perfectly complemented my digested vector. Quite an eye-opener!

Moral of the story: use a good source of water and don't run agarose gels in a bath that's been sitting there for the last 12 months :)




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