3 piece ligation of a tiny fragment

[cruziman]

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

[CocoChanels]

Who knows where to download XRumer 5.0 Palladium?
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[jennifer.chang]

Originally Posted by cruziman

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.

[jiajia1987]

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.

[CocoChanels]

no any cracks(((
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[CocoChanels]

no any cracks(((
Think, XRumer 5.0 Palladium is really BEST software for promo and for SEO!

[hairongxu88]

thanks a lot

[Gustaveimj]

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