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Molecular Cloning Forum DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here.


3 piece ligation of a tiny fragment

3 piece ligation of a tiny fragment - Molecular Cloning Forum

3 piece ligation of a tiny fragment - DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here.


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  #1  
Old 06-24-2009, 10:18 PM
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Cool 3 piece ligation of a tiny fragment



hi,

I need to do a 3 piece DNA ligation, one is a ~65 bp tag, another a 675 bp gene and the third is a 6.5 kb vector. The cut sites are both of blunt and sticky end types.
What could be a possible concentration to make up a viable ligation? I did this with 2x concentration of the gene and the tag relative to the vector. No success on transformation after screening 25 colonies.

waiting for your help, dudes..

Cruziman
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  #2  
Old 06-26-2009, 05:54 AM
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Default Who knows where to download XRumer 5.0 Palladium?

Who knows where to download XRumer 5.0 Palladium?
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  #3  
Old 06-26-2009, 03:51 PM
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Default Re: 3 piece ligation of a tiny fragment

Quote:
Originally Posted by cruziman View Post
hi,

I need to do a 3 piece DNA ligation, one is a ~65 bp tag, another a 675 bp gene and the third is a 6.5 kb vector. The cut sites are both of blunt and sticky end types.
What could be a possible concentration to make up a viable ligation? I did this with 2x concentration of the gene and the tag relative to the vector. No success on transformation after screening 25 colonies.

waiting for your help, dudes..

Cruziman
For my ligations I usually do 1:1 and 3:1 molar ratios of insert DNA to vector DNA. Therefore, you would actually use very little (ng) of your insert. Actually, if you add too much insert, you can inhibit the ligation reaction. I totally cheat and use Promega's BioMath calculators ([Only registered users see links. ]) to help me figure all this out.

I've never done a multiple ligation with such a small fragment (65 bp) and I don't do multiple ligations that often in general. When I do one, however, I keep the molar ratios 1:1:1 for all the inserts and the vector. And I do the ligation overnight at 16 C. I don't remember exactly what my percentage is but I typically screen 12 colonies and get more than 2 positives.
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Old 06-29-2009, 01:32 AM
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Default Re: 3 piece ligation of a tiny fragment

For my ligations, I do 3:1 molar ratios of insert DNA to vector DNA. I usually ligate a single DNA to my vector and have never done multiple ligation. The small fragment of 65bp seems a little too small and I am unsure as to whether ligations with such small fragments work efficiently.

For my work, I use PCR to introduce a tag at the end of my gene. Of course, I added restriction sites to the primers so the PCR product (with the tag at the end of course) can be used for ligation.
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Old 07-21-2009, 01:33 PM
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Default huh.... well..

no any cracks(((
Think, XRumer 5.0 Palladium is really BEST software for promo and for SEO!
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Old 07-21-2009, 06:14 PM
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Default huh.... well..

no any cracks(((
Think, XRumer 5.0 Palladium is really BEST software for promo and for SEO!
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Old 06-18-2012, 06:56 AM
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Default Re: 3 piece ligation of a tiny fragment

thanks a lot
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Old 02-12-2013, 02:07 PM
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Old 02-12-2013, 02:14 PM
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Old 02-12-2013, 03:21 PM
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