I have eluted plasmid backbone after double digestion using Qiagen spin column. When I ran my samples on the agarose gel I could see a single band of intensity almost comparable to 500 bp ladder band. But when I am measuring oD of the same sample (2ul) using nanodrop I am getting concentration values varying from 3 to 7 ng/ul...nowhere close to what I see in the gel. I have to setup a ligation reaction with the same. Can anyone please tell me what I may be missing or how to proceed.
Hi!
When I have to proceed with a ligation, I usually use the formula presented in the TA Cloning manual, where you consider the lenght and the amount (in ng) of your plasmid, the lenght of your insert and in this way you obtain the amount (in ng) of the insert to use. It may help you! Additionally, I want to suggest you to do, if possible, 2 or 3 ligation using different quantity of your plasmid!
Good luck!
What kind buffer did you elute the DNA from the column? It is better to use the same buffer as Blank when you measure the DNA con using Nano Drop. Since you could see the band on the agarose gel, just trust the band on the gel (seeing is believing!). I set up the ligation reaction as following:
plasmid backbone: 10-30ng
insertion fragment: 3x molecule number relative to plasmid molecular number
buffer and T4 ligase( we routing use NEB T4 DNA ligase 1h @RT)
Also need set up only cut plasmid ligation as control.
Good luck!
The gel is more accurate than the Nanodrop. If the 500 bp band is what you're looking for, purify it and proceed. If accurate concentration measurements are important, you can try loading a few lanes of a dilution series of something you trust you know the concentration of (with a mw in the same neighborhood), but doing 2-3 ligations as suggested above is probably a lot faster than nailing down a precise concentration. If this is a straightforward ligation, the transformation will clear things up and you'll get more used to how quantities look with practice. You can also check ladder and Lambda phage digest images from vendors' websites for approximations of quantity/appearance.
Thanks Giulia, Peng and Melissa for your valuable suggestions.l will try different ligations. @ Peng I had eluted the dna in Autoclaved milli Q n used the same as blank.
Hi,
I have a doubt. You said you eluted backbone. Then you ran a gel. What you loaded in gel? Insert or Vector backbone? I assume insert.
I have two suggestions for measuring the DNA concentration.
1. Pico green assay: Pico green is a dsDNA binding dye, which can measure DNA in pico quantities. The kit is available from Lifetech.
2. UV quantification: Prepare Sample dilutions like 1:250, 1:500 and 1:1000 and read on a UV spec. Use this only when you have sufficient sample volumes.
Last but not least, always remember that amount of DNA in a ligation reaction should not exceed 200ng (including vector and insert DNA).
Hope this helps...
Good luck... :)
I always set up my ligation reactions with this formula: http://www.promega.com/a/apps/biomath/index.html?calc=ratio
It's really easy to use, but make sure you don't add too much plasmid to the reaction because it won't work (100 ng in total - vector and insert- should be okay!)
Hope you find this useful,
Cheers
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