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help for large-scale plasmid purification


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#1 jimlulu

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Posted 05 October 2004 - 09:02 AM

today i have purified plasmid from 500ml cultural media, i add 10ml solution 1and mix equably before add 20 ml solution 2, but in this time ,i can't make the liquid clear ,and there are always a group of e.coli like to be pack by DNA. Is the solution not enough or other reasons?

i want to purified plasmid for DNA vaccine, can you give me any protocol to get 10-20mg plasmid ? thank you very much

#2 SVTX

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Posted 05 October 2004 - 02:07 PM

Hi,

I have used Qiagen's Maxiprep for isolating BACs, which are single copy, quite successfully. I have got up to 1.5 mg of DNA. I was using only 300 ml o/n culture. You might be able to get better yield since you are using more starting material plus, I presume your's is multi-copy plasmid. You may use the reagents that come with the kit but avoid the columns and instead just ppt the DNA using isopropopanol to even better the results.

Like in minipreps, you may not see a clear solution after the lysis when you are dealing with maxipreps.

Good luck!

SV

#3 tfitzwater

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Posted 12 October 2004 - 04:35 PM

1. Solution I: Resuspend a 500 mL cell pellet in 10 mL of Solution I without lysozyme. Then add 10 mL of Solution I with 8 mg/mL lysozyme or Solution I with RNase to the resuspended pellet. Mix and incubate on ice for 15 minutes. Option: add Solution I without lysozyme, resuspend the pellets and store at 20C for up to 5 days. Vortex the cell pellet till completely resuspended or use a plastic pipette tip to mix and resuspend. A pipette tip is more gentle and may prevent shearing of chromosomal DNA. Some strains of E. coli are mucoid and resistant to resuspension by vortexing. Partial lysis of the top of the cell pellet may prevent resuspension of the bottom of the pellet, so mixing is required.

2. Solution II: the use of ice-cold Solution II prevents sudsing. Invert the tube gently until the mixture is clear, generally 5-7 times. Incubate at room temperature for 10 minutes or until the solution is clear. If high cellular concentrations or plasmid DNA density makes this step slow, reduce the volume of cells used to prepare the pellet in step 1. A slight trace of cellular turbidity is permissible. Do not vortex mixtures containing Solution II, as this will shear chromosomal DNA which will then contaminate the plasmid prep. Some authors allow a 30 minute incubation at room temperature

3. Solution III: a curd-like precipitate will form when Solution III is added. If the cell pellet was particularly large, increase the incubation time of Solution III to 10 minutes. The white slurry is primarily composed of the potassium salt of dodecyl sulfate plus chromosomal DNA and cellular debris. Plasmid ccc DNA is in the clear supernatant. If necessary, the reaction can remain on ice for over 60 minutes.

#4 Makedonce

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Posted 13 October 2004 - 07:42 AM

I don't know why would you like to make the liquid clear after adding solution 2??? At this point the bacteria should be lysing and you should get something that looks like a goo....slimy and sticky.
What I found to be working is to spin your culture and add 100 mls of solution 1. Completly resuspend the pellet and spin it down again (4000rpm for 10 minutes at 4C). Discard the solution and add your desired volume of Solution 1 (I use 12 mls but adjust the protocol to your needs). COMPLETLY resuspend the pellet again. Then add solution 2 (I add 16mls) and let it sit on your bench for few minutes. The add solution 3 (I add 12 mls) and gently swirl and keep on ice for 10 minutes.
After a spin at 12000rpm for 20 minutes at 4C, all the cell debris will be pelleted but few "chunks" will be floating in the supernatant.
At this point you want the clear liquid. While pouring the supernatant into a fresh tube filter it through a sterile gauze. This will help you get rid of most of those stubborn "chunks" floating around:)

Basicaly this is straight forward alkaline lysis with addition of that extra wash with solution 1.




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