I'm trying to transform GFP plasmids to E. coli and clone them.
We tried it but the concentration is too low, like 100ug/ul.
We use 50ug of GFP plasmids for each experiment and the concentration is like 2800ug/ul.
Is there a good kit for extracting very high concentration of DNA?
But then, the problem is we don't have a centrifuger that can work with high volume of tubes.
Our machines can only deal with tube volumes less than 50ml.
Besides, we have many kits that are left unused for a few years...
So I need better options that are affordable and easier to apply...
any suggestions?
Dear Hyunbae
which vector are you using?
Generally i'm using the EZNA MINI KIT II of Omega BIotek which is able to provide higher yields respect than standard kits.
You can find some examples on my blog ProteoCool
https://proteocool.blogspot.com/
ProteoCool n°12
(E.Z.N.A HP MINI KIT II: MIDI KIT YIELDS from a MINI KIT format) available at PAGE6
However you can reach concentrations around 700-1000mg/ul but more than 2000 it is probabily not possible.
I think that the only way to acheive it is perform a pool of several mini kit and concentrate the DNA using or:
- A speed vac instrument
- perform a ethanol or isopropanol DNA precipitation in 1.5ml eppendorf (as happen in the final step of some MIDI KIT) and resuspend in small volume of water.
good luck
Manuele
Dear Hyunbae
which vector are you using?
Generally i'm using the EZNA MINI KIT II of Omega BIotek which is able to provide higher yields respect than standard kits.
You can find some examples on my blog ProteoCool
https://proteocool.blogspot.com/
ProteoCool n°12
(E.Z.N.A HP MINI KIT II: MIDI KIT YIELDS from a MINI KIT format) available at PAGE6
However you can reach concentrations around 700-1000mg/ul but more than 2000 it is probabily not possible.
I think that the only way to acheive it is perform a pool of several mini kit and concentrate the DNA using or:
- A speed vac instrument
- perform a ethanol or isopropanol DNA precipitation in 1.5ml eppendorf (as happen in the final step of some MIDI KIT) and resuspend in small volume of water.
good luck
Manuele
According to the plasmid isolation, the yield will depend on the copy number of your plasmid. Anyway, we have used ACCuPrep Nano-Plus plasmid Mini Extraction kit.Quit easy and concentration of plasmid around >1000 ng/ µL.
Centrifuge is doesn't much care, but find the plasmid category of your bacteria either low or high no of plasmids.
https://eng.bioneer.com/20-k-3030-cfg.html.
https://eng.bioneer.com/20-k-3122.html
I suggest Maxi prep kit from qiagen. it should give you around 2 ug in 1 ml.
I think that you must be doing something wrong, because you should get plenty of colonies after transformation of E. coli with even less than 100 µg/µl.
Check the quality of the plasmid DNA and use the appropriate genetic marker.
there are plenty of protocols to increase the concen. of plasmid. we used sodium-acetate to increase the concentration of isolated plasmids as much as possible. However, if i were you, I would use Maxi-prep and also sodium-acetate to increase the plasmids' conc.
The concentration of the plasmid can be increased by reprecipitation with alcohol
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