Plasmid Extraction - Middle Tennessee State University：質(zhì)粒提取-田納西中部州立大學(xué)

1、Genetics 3250 Sequence AnalysisCahoon Genetics 3250 LabMiddle Tennessee State UniversityChloroplast Genome Project Week 1In plant cells, DNA can be found in three organelles nuclei, chloroplasts, and mitochondria.nucleuschloroplastmitochondrionmitochondrionchloroplastchloroplastThe chloroplast is th

2、e site of photosynthesis in plant cells. Its genome and its process of gene expression is very similar to those found in bacteria.Maier et al. 1995, J. Mol. Biol 251:614-628The chloroplast genome can be imagined as a circular piece of DNA about 120,000 140,000 bases in size with approximately 100 ge

3、nes. This diagram represents the corn chloroplast genome which was completed in 1995.You and your genetics class colleagues are attempting to sequence the chloroplast genome of tall fescue (Festuca arundinacea). Intact genomes are generally too large to sequence. To get around this problem a genome

4、must be cut into lots of small manageable pieces. This semester, two independent study students have isolated the chloroplast genome, mechanically sheared the DNA into small pieces and then placed them into plasmid cloning vectors for sequencing.genomePieces put into plasmid vectorsShearingforceGeno

5、me cut into thousands of tiny random piecesThese plasmids are then grown inside bacteria that will produce the millions of copies that are required for manipulation and sequencing. Today you will extract a plasmid from a bacterial culture. Next week we will use this plasmid to set up a sequencing re

6、action.Plasmid DNA Extraction Plasmid ExtractionGoal extract the plasmid DNA from a liquid bacterial culture.Materials· Resuspension Buffer (P1)· Lysis Buffer (P2)· Neutralization Buffer (N3)· Wash Buffer (PE)· Spin Column· Elution Buffer (EB)· Collection tubesIntr

7、oductionToday you will use a commercial kit (Qiagens Miniprep) to extract and purify plasmid DNA from your bacterial culture. Plasmids are small circular pieces of DNA that are maintained and replicated separately from the bacterial cells genome. Procedure1. Pipette or pour 2.0ml of culture into a c

8、olored microfuge tube.2. Pellet cells for 1minute at full speed.3. Pour off the supernatant (growth medium) and blot the open tube against a paper towel. Resuspend your pellet in 250l of Resuspension Solution (P1) by vortexing until you no longer see a pellet.This step removes the salty growth mediu

9、m and resuspends in a buffered solution which will aid the extraction.4. Add 250l of Cell Lysis Solution (P2) and mix by rocking the tube in your fingers. The solution should become mostly clear and viscous.The cell lysis solution contains a strong detergent (Sodium Dodecyl Sulfate) and base (Sodium

10、 Hydroxide) which will lyse the cells and denature proteins and membrane components so the cell will spill its contents. After this step you have a mixture of every type of molecule found in the cell. The challenge is to purify the DNA away from everything else (protein, lipid, carbohydrate, and ins

11、oluble debris). 5. Add 350l of Neutralization Solution (N3) and mix by rocking the tube. A white precipitate should form.The solution is Potassium Acetate. The acetic acid neutralizes the NaOH rendering the SDS insoluble. When the SDS leaves solution it takes most of the protein and lipid with it.6.

12、 Centrifuge at full speed for 10 minutes.The purpose of this step is to pellet the white precipitate of SDS, protein, and cellular debris.7. Label a minicolumn with your clone number.Pipette the supernatant from step 6 into column. Centrifuge for 30-60 seconds. Remove the column, pour off the flow-t

13、hrough and replace the column in the tube.The minicolumn contains microscopic charged glass beads. As the aqueous supernatant passes through the glass matrix in the column, certain hydrostatic molecules (like DNA) will stick to the beads. Most of the residual proteins and carbohydrates will pass by

14、the beads and end up in the flow-through which is discarded.8. Add 750 l of Wash Solution (PE) to the column and centrifuge for 30-60 seconds. Remove the column, discard the flow-through and replace the column in the tube.Wash solution is a mixture of solutes and alcohol. This mix contains just enou

15、gh ethyl alcohol to prevent DNA from solubilizing and leaving the glass bead matrix. Almost all other charged molecules will become soluble and wash off of the glass matrix.9. Centrifuge the column in the collection tube for 1 minute to remove any residual wash buffer.10. Label a new microcentrifuge tube with your clone number and name. Place the column into the new tube.11. Add 50l of Elution Buffer (EB) or Sterile Water to the column. Let stand for 1 minute and then centrifuge for 1 minute.The interaction between D