Plasmid Dna Preparation

Plasmid DNA Preparation

Abraham Mikru University of Helsinki Department of Applied Chemistry and Microbiology 13/12/08

12/19/08

Abraham Mikru University of Helsinki, Dec 13, 08

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Overview  Similar general strategy to genomic DNA preparation  Grow the bacteria with plasmid in broth  Harvest cells and prepare extract  Remove protein and RNA  Concentrate DNA with Ethanol PPt  Crucial difference is the need to separate plasmid DNA from Chromosomal DNA 12/19/08

Abraham Mikru University of Helsinki, Dec 13, 08

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Separation of plasmid from Chromosomal DNA  Purpose is to use the plasmid as cloning vector  Even minute amounts of contaminating chromosomal DNA is undesirable  Several separation methods  Alternatively or in combinations  All exploit physical differences between the two  Size  Conformation- linear or circular

12/19/08

Abraham Mikru University of Helsinki, Dec 13, 08

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Separation Based on Size

 Size fractionation done usually at extraction phase  Careful lyses procedures to avoid breakage of chromosomal DNA  Controlled cell lyses by combination of EDTA +Lysozyme in sucrose  Prevents cell from sudden bursting  Spheroplasts are formed with partially digested wall  Lyses induced by non-ionic detergents such as Triton X-100 (ionic detergents SDS, breaks chrom DNA)  Even when broken, fragments remain larger than plasmid DNA  Easy to remove by centrifugation  Some fragments may remain and must be removed  If plasmids are too large they may also be removed with cell debris- need to consider alternative procedures 12/19/08

Abraham Mikru University of Helsinki, Dec 13, 08

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Separation Based on Conformation         

Most plasmids exist in supercoiled state Covalently closed circular (CCC) DNA Due to partial unwinding by topoisomerase Occurs during replication A nick in on of the strand lead to relaxed Open circular (OC) DNA conformation Supercoileds are easy to separate from OC Two methods:- Alkaline Denaturation Density gradient centrifugation

12/19/08

Abraham Mikru University of Helsinki, Dec 13, 08

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Alkaline Denaturation  Narrow pH range at which OCDNA is denatured but not CCCDNA  The pH adjusted to12 -12.5 by NaOH  The two strands in OCDNA separated  Add acid and the two strands tangled  Pellet out tangled mass by centrifugation  Plasmid DNA remains in the supernatant  Advantage in case of lyses by SDS and neutralization by Na acetate  Proteins and RNA can also be denatured and precipitated- easily removed by centrifugation  Further purification by organic extraction or column chromatography may not be needed 12/19/08

Abraham Mikru University of Helsinki, Dec 13, 08

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EtBr – CsCl Density gradient centrifugation  Specialized version of equilibrium or density gradient centrifugation  Density gradient produced by high speed centrifugation  A solution of CsCl  Macromolecules segregate in the column based on their buoyant density – form distinct bands  Buoyant density of DNA is 1.7g/cm3  Migrate to the corresponding density in column of CsCl  Proteins are lighter and floats at the top  RNA forms pellet at bottom  Thus DGC is an alternative to organic extraction or column chromatography for DNA purification 12/19/08

Abraham Mikru University of Helsinki, Dec 13, 08

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DGC in the presence of EtBr             

Used to separate supercoiled from non-supercoiled DNA EtBr intercalates between adjacent base pairs Cause partial unwinding of duplex DNA The unwinding reduces buoyant density by ≈0.125g/cm3 for linear DNA Supercoiled DNA with no free ends has little freedom to unwind Only limited amount of EtBr is bound BD increases only by 0.085g/cm3 Thus in EtBr-CsCl DGC supercoiled DNA forms a distinct band below that of linear or OCDNA Position of DNA bands seen under UV The pure plasmid DNA removed by puncturing side of tube with needle EtBr is extracted from the plasmid DNA with n-Butanol The CsCl removed by dialysis This is a very efficient way to get 100% pure plasmid DNA

12/19/08

Abraham Mikru University of Helsinki, Dec 13, 08

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Plasmid Amplification  Plasmids make only a small proportion of total DNA in a bacterial cell.  The yield of plasmid from culture too low  Plasmid amplification offers means of increasing the yield  Aim is to increase copy number  Some multicopy plasmids (with 20 or more) replicate in the absence of protein synthesis  Chromosomal DNA doesn’t replicate under this condition  Grow bacteria for plasmid DNA preparation  When cell density reaches some level add chloramphenicol to block protein synthesis  Incubate further 12 h to allow plasmid replication  Plasmid copy number of several thousands may be achieved

12/19/08

Abraham Mikru University of Helsinki, Dec 13, 08

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