Team:Debrecen-Hungary/protocols/midiprep
From 2010.igem.org
Scientific BackgroundOverviewProcedure for High Copy Number Plasmids Materials1. Sample material: 30 ml E. coli culture, transformed with a high copy number plasmid Harvest cultures at a density between 2.0 and 6.0 A600 units per ml bacterial culture 2. Media: The isolation method is optimized for cultures grown in LB media; other rich media may require increased volumes of Suspension-, Lysis- and Neutralization Buffer, and an additional wash step 3. Plasmid size: The isolation procedure is suitable for all plasmid sizes; lysates of larger constructs (up to 100 kb) should be cleared by filtration to avoid shearing 4. Suspension Buffer/RNase A: To dissolve the lyophilized enzyme in Suspension Buffer, pipet 1 ml of Suspension Buffer (bottle 1, black cap) into the glass vial containing the lyophilized RNase (bottle 2, black cap). Reinsert the rubber stopper and invert the vial until all lyophilizate (including any that might stick to the rubber stopper) is dissolved. Transfer the dissolved enzyme back to the Suspension Buffer bottle (bottle 1). This is enough working solution for 60 Midi preps (isolation of up to 100 μg plasmid DNA/preparation) 5. If preparing aliquots of the working solution, remember that the final concentration of RNase A in the working solution must be 100 g/ml. Reconstituted buffer is stable for 6 months is stored properly (+2 to +8°C) 6. Neutralization Buffer: Before starting, cool it down to 4°C 7. Elution Buffer: Before starting, warm up the buffer to 50°C 8. Ethanol: Use 70% ethanol. Before starting, cool it down to 4°C Procedure1. Centrifuge bacterial cells from 30 ml culture grown in LB medium by centrifuging for 10 min at 5000 rpm,4°C
2. Add 4 ml Lysis Buffer to the suspension and mix gently by inverting the tube 6 times.
Incubate 2-3 min at room temperature.
To avoid shearing genomic DNA, do not vortex the suspension in Lysis Buffer. To prevent release of chromosomal DNA from the cell debris, do not incubate for more than 5 minutes
6. Load the cleared lysate from step 4 onto the equilibrate column.
Allow the column to empty by gravity flow.
Discard the flowthrough
11. Divide the 6 tubes into two groups. Wash the plasmid DNA with 1,5 ml chilled (+4°C) 70% ethanol in the first tube and afterwards wash the other two with the same ethanol. Wash the other group (3 tubes) the same way.Centrifuge 10 min at 15000×g (rcf), +4°C.
Carefully remove ethanol from the tube with pipet tip.Air-dry the plasmid DNA pellet for 10 min.
12. Carefully redissolve the plasmid DNA pellet in 100 μl TE buffer.
Notes & troubleshootingReferences1.Birnboim, H.C. and Doly, J. (1979) A rapid alkaline lysis procedure for screening recombinant plasmid DNA. Nucl. Acids Res. 7, 1513-1522 2.Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd. Edition, Cold Spring Harbour Laboratory Press 3.Ausubel, F.M. et al. (eds.) (1991) Current Protocols in Molecular Biology, Wiley Interscience, New York Links[http://www.youtube.com/user/debrecenigem2010#p/u/18/4JyHe5gxihU Video I] [http://www.youtube.com/user/debrecenigem2010#p/u/17/Jots5XV6ZOA Video II] [http://www.youtube.com/user/debrecenigem2010#p/u/16/NRQ_z11mGnw Video III] [http://www.youtube.com/user/debrecenigem2010#p/u/15/Iq7bYjeeSf0 Video IV] [http://www.youtube.com/user/debrecenigem2010#p/u/13/z2Sav6GKSgs Video V] [http://www.youtube.com/user/debrecenigem2010#p/u/12/HbR3qZ5Stxk Video VI] [http://www.youtube.com/user/debrecenigem2010#p/u/11/ljafO21XPow Video VII] [http://www.youtube.com/user/debrecenigem2010#p/u/10/JWGGDGPiz1U Video VIII] [http://www.youtube.com/user/debrecenigem2010#p/u/14/dxv63-rBc7E Video IX] [http://www.youtube.com/user/debrecenigem2010#p/u/8/d2_HV8Mx0go Video X] [http://www.youtube.com/user/debrecenigem2010#p/u/9/0AFgNjVbFGg Video XI] |