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ligation help


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3 replies to this topic

#1 netnus

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Posted 17 September 2004 - 05:59 AM

I want to ligate a 500bp DNA into the vector pET9-a. I had already double digested the vector plasmid and the insert (500bp DNA). The digested plasmid was also purified by extraction from the agarose gel. The ligase I use is T4 ligase from Fermentas. I follow the protocol they give us.After ligation is done, I run a gel but can't see anything. Since I don't know the concentration of the plasmid and the insert. I make 20 microliter sample with 8 microliter plasmid and 10microliter insert.
The complete protocol is below:
8 microliter vector
10 microliter insert DNA
2 microliter 10X T4 ligation buffer
0.4 microliter T4 ligase.
Incubate at 22 degree for 1 hour;
Inactive T4 by heating the reaction at 65 degree for 10 min.

Bs: I load 10 microliter of the mixture while I run the gel?

Can anybody tell me what I can do ? Thank you very much!!

netnus

#2 biotech

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Posted 17 September 2004 - 10:52 AM

Well, if you don't see anything on your gel, you either have insufficient amount of DNA to see, or forgot to add ethidium bromide to the gel. You write the volumes of your vector and insert but what is their concentration? Usually, you cannot see more than <100 ng of DNA on an agarose gel, maybe that's the reason?

#3 pcrman

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Posted 17 September 2004 - 05:28 PM

Do you know how much plasmid you initially added for digestion? You should have an idea how much plasmid and insert are used in the ligation reaction. After the ligation, load 4 ul onto a gel, you should be able to see something on the gel. If ligation is good, you will see circular plasmid (two bands). I agree with biotech, those he mentioned are potential problems.

#4 netnus

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Posted 17 September 2004 - 07:36 PM

Well, if you don't see anything on your gel, you either have insufficient amount of DNA to see, or forgot to add ethidium bromide to the gel. You write the volumes of your vector and insert but what is their concentration? Usually, you cannot see more than <100 ng of DNA on an agarose gel, maybe that's the reason?

Firstly thank you very much for your reply!
I use fluorometer to detect the concentration of purified plasmid. It is very low only 15-20ng/ul. The digested insert isn't purified after the double digestion since I loss almost all the DNA while purification. Do you think it is necessary to purify the insert firstly before ligation?
Can I use the ligated mixture for transformation even though I can't see the bands in the gel?

Hope to see your reply soon!
Thanks a lot!




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