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sequential cloning of multiple PCR products


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3 replies to this topic

#1 Curtis

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Posted 01 August 2011 - 10:10 AM

I got 8 PCR products that need to be sequentially ligated to each other in order to make a full-length genome.

Each PCR product shares the same RE site with the next PCR product (see attached paper,I'm following the same protocol)

the question is, after ligating the first 2 PCR products, do I need to gel purify the ligation? As we know after gel purification we normally lose a lot of DNA, so if I'm supposed to use the purified ligated DNA for the next digestion and ligation then I will lose all the DNA before I reach the final PCR product!!!!

So how?

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#2 bob1

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Posted 01 August 2011 - 03:23 PM

If you are cloning into a vector, just ligate the vector closed and then grow it up after each product, that way you will always have heaps of DNA for future preps, as well as backups in case something goes wrong.

#3 Curtis

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Posted 02 August 2011 - 01:19 AM

Thanks Bob1, as usual,

But I'm not cloning into a vector. just ligating the PCR products with each other.

How to make sure if the two PCR products ligate at all?

Can I see the ligated band if I run the ligation on the gel? I have never loaded the ligation mixture on the gel.

Wouldn't the bands be very faint? because for ligation we normally us low amount (maximum 100ng) of DNA....if the fragments don't ligate I'm screwed!

Edited by Curtis, 02 August 2011 - 01:21 AM.


#4 phage434

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Posted 02 August 2011 - 04:01 AM

I don't know how you have set up your restriction sites. If they are all incompatible sites, then it should be possible to ligate the parts all at the same time. This would be difficult, with many possible things that could go wrong, and with no easy way to debug what is happening, however. I think you would be much better off to ligate and clone shorter fragments, amplifying the amounts of DNA at each stage with minipreps, and allowing sequencing of intermediate results. In the long run, I think this would be easier.

You can visualize ligation products. Use normal (not quick) ligase buffer. Heat kill the ligase. Run a normal gel. A useful debugging technique is to test ligate pairs of parts, which are supposed to ligate, and look for the double length product. You can do this for each pair, and figure out what is ligating and what is not. Your test ligations need not be at low concentrations, although your final circularization reaction should be.




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