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Insert & vector too dilute to perform ligation


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#1 tmaz

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Posted 09 December 2013 - 06:37 AM

Hi,

 

All ligation protocols which I've found requires 10-20 ul reaction volume and 50-100 ng of vector or insert. When I purify the digested insert or vector from the gel, the concentration is always about 5 ng/ul. So It's impossible to do the ligation in 10-20 ul volume. 

 

Can you help me with this? Can I do the ligation in bigger volume? How about the amount of DNA..could I use less insert or vector? I would like to use 6:1 or 3:1 molar ratio of insert to vector.

 

Thanks!



#2 phage434

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Posted 09 December 2013 - 11:09 AM

The articles are  wrong. You WANT low concentrations. High concentratios give rise to multimers, which do not transform. You want low concentration, which favors the intramolecular circularization necessary to produce active plasmids. The optimum depends on plasmid size, but is below 20 ng/ul for normal sized plasmids. You can calculate it -- you want the concentration of the other end of the plasmid to be high relative to the end of a different fragment of DNA.

 

You can concentrate AFTER ligation if you need to, but high competence cells make that unnecessary.  Don't transform ligation product with more than about 5% of the competent cell volume.



#3 tmaz

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Posted 11 December 2013 - 12:50 AM

Thank you for the answer!

 

I managed to get my vector to concentration of 25 ng/ul and did the the ligation in 20 ul volume using 50ng of vector and 3:1 molar ratio of insert to vector. I tranformed 2 ul of ligation reaction to 50 ul of CaCl2 competent E.coli cells and did a heat shock transformation. I plated only 15 ul of cells resulting to 0 colonies. I now plated 10x volume and I'm waiting fingers crossed to see some colonies tomorrow. My competent cells are fine since the plain vector control gives colonies.



#4 phage434

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Posted 11 December 2013 - 01:37 PM

Cue the often repeated statement. Your "competent" cells are NOT fine. CaCl2 competent cells are fine for tranforming a miniprep plasmid sample. The are NOT fine for cloning applications. You need good competent cells with 10^8 cfu/ug or so competence. You can't get there with CaCl2 transformation. Buy competent cells (easiest) or use a better protocol and measure the competence.






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