large plasmid extraction? - how? (Dec/07/2007 )

hi

im trying to extract and clean a plasmid ~140kb from DH10B.
i use the clasical protocols (alkali) with the three solution and the ethanol precipitation.
however my plasmid does not come out clean enough (it has a lot of RNA, cell debris, proteins, salts...). I need it more clean!
Any suggestion about kits or other methods you have used?
thanks in advance...

tasos

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try qiagen large construct kit

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You could also phenol/chloroform extract. Your major diffficulty will be the mechanical fragility of plasmids of this size. Don't vortex and pipet carefully with wide bore tips.

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Quite right, the key to this work is tender loving care (TLC).

Important places to pay attention is

Growing conditions
Large plasmids don't grow well. So it improtant to grow your cells in a rich media. A slow growth rate can sometimes help, this can be achieved by growing the cells at a lower temperature. LB you will find isn't a good enough medium for very large plasmids. Yeilds are poor. Consider changing to a better medium like SOC or terrific broth, which improves yeilds and plasmid integrity.

When adding solution 2 (alkaline lysis solution).
- the cell pellet is now resuspended in solutionI. Add solution II.
I tend to add more solution II when working with large BAC (50kb and above) with a proportional increase of solution III to compensate. Swirl gently, once and leave the container to stand for 5mins. The ideal container for this work is one which is wide. Tall containers form columns and mixing between the two solutions only occurs at the interface, thus a wide container is desireable. The ideal situation is where the two solutions mix on their own accord with no outside influence.

Cooling
Cooling solutions on ice prior to centrifuging seems to help too. Must be the low yeilds

Pipetting,
I have found the best pipette is actually a plastic dropper with the end cut of just a little.

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Hi,

to extract 140kb plasmid, you can try as suggested in
http://www.bio.net/bionet/mm/microbiology/...ary/001083.html

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i can recommend you the protocol i use :

2ml culture with appropriate selection
do not vortex, pipett always gently, do not freeze thaw (but 2cylces don't affect prep quality)
put in eppi. Spin 1' 13000g
resuspend cells with 0,3 ml solution 50mM Tris HCL pH :8,0 ; 10mM EDTA ; 100 µg/ml RNase (P1 prep quiagen)
add 0,3 ml d’une solution 0,2 N NaOH, 1%SDS, homogenize gently.
Let incubate 5-10' RT
add 0,3 ml of solution Na acétate 3M homogenize gently during addition.5-10' on ice
spin 10 min 13 000g R.T.
Put 0.8ml IprOH and add the supernatant of the spinhomogenize slow by inverting tube.
5-10' on ice and spin 15' RT 13000g
wash by 0.5ml ethanol 70% twice
spin for 5' at 13 000g
Let dry 5 10 min R.T
Dissolve in 40 µl TE.
store at 4°C never freeze

You may also have a look at this thread

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