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How to increase the concentration insert or vector or no cut plasmid - How to increase the concentration insert or vector or no cut plasmid (Apr/05/2009 )

Hi everyone, :(
I would like to inform how I can concentrate my plasmids that carry a low copy number before i use them in restriction digestion and then in cloning , because both the plasmids after harvest them by miniprep are very low concentration and that don't help me for successfully finish the ligation step.
Any suggestion please to concentrate my insert or vector or no cut plasmids ?
Thanks
David

-dawood-

Hey,

For low copy number plasmids:

I inoculate 6 separate tubes (3 mL each) and perform minipreps for each one of them. In the end, I sequentially resuspend in 30 ul water. What you do is that you add 30ul to the first tube, vortex, spin and aspirate this 30ul andput it in the 2nd tube and do this with all 6. You end up with good concentration (I use about 3 ul per digestion).

For low concentration of digested fragments:

1. Put multiple digestions and pool at the gel elution stage. If the yield is still low, concentrate using speed vac.

Hope it helps.

Best,
TC

-T C-

T C on Apr 5 2009, 10:10 PM said:

Hey,
Sorry i didn't understand your procedure experimentaly can you explain that again with details
Thanks again

For low copy number plasmids:

I inoculate 6 separate tubes (3 mL each) and perform minipreps for each one of them. In the end, I sequentially resuspend in 30 ul water. What you do is that you add 30ul to the first tube, vortex, spin and aspirate this 30ul andput it in the 2nd tube and do this with all 6. You end up with good concentration (I use about 3 ul per digestion).

For low concentration of digested fragments:

1. Put multiple digestions and pool at the gel elution stage. If the yield is still low, concentrate using speed vac.

Hope it helps.

Best,
TC

Attached File

-dawood-

Hey,

Just incoluate 6 or more tubes with the same culture. Proceed with miniprep with all of them in the normal way. In the end, you would have one vial of dried DNA from each miniprep. Now instead of resuspending each of them in 30 ul water, add 30 ul to first, resusppend that DNA and take out this and transfer to second tube and so on (do it sequentially).

So instead of having all vials with 30 ul DNA in each, you would have the DNA from all the vials in 30 ul total and therefore it would be concentrated.

Hope this makes sense :)

and it helps.

Best,
TC

-T C-

You can try to concentrate it by using glycogen.
This is the protocol I use from fermentas:

1. Add 1/10 volume of 3 M sodium acetate (or 2 M sodium chloride, or 5 M ammonium acetate) to DNA solution.
2. Add Glycogen to a final 0.05-1 g/l concentration. Use up to 1 l of Glycogen per 20 l of the solution.
3. Add 1 volume of isopropanol (or 2.5 volumes of ethanol) to the solution. Mix gently but thoroughly. Ethanol is recommended for precipitation of smaller than 200 b/bp RNA/DNA fragments.
4. Incubate the mixture at -20C for up to 60 min, or at -70C for 30 min.
5. Centrifuge the mixture for 10-15 min at 10,000 rpm. Discard the supernatant.
6. Rinse the pellet with cold 70% ethanol. Air-dry the pellet.
7. Dissolve DNA in Water, nuclease-free or TE buffer.
8. Dissolve RNA in DEPC-treated Water.

-molgen-