Team:Arizona State/Lab/Protocols/Extraction

 

Ethanol precipitation

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This protocol purifies / concentrates a DNA sample.

1. Measure out 2-3 volumes 100% ethanol into sample.
2. Mix solution by pipetting up and down or by inverting the tube several times.
3. Incubate at -20C for 12 hour, or for 20 minutes to 1 hour at -80C.
4. Centrifuge at maximum speed, at 4C, for 30 minutes. Discard supernatant.
5. Airdry for 15 minutes to remove excess ethanol.
6. Resuspend pellet in a 1.5 ml microcentrifuge tube. Be careful of the pH of the water used, which can vary widely. The nucleic acid should be suspended at a neutral pH.

Gel extraction

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Qiagen Miniprep:

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The following protocol is applicable to the [http://www.qiagen.com/products/plasmid/qiaprepminiprepsystem/qiaprepspinminiprepkit.aspx QIAprep Spin Miniprep Kit], using micro-centrifuge:

1. Add 1.5 mL of liquid culture to microcentrifuge tube and centrifuge at 13,000 rpm for 1 minute. Discard supernatant.
2. Resuspend pelleted bacterial cells in 250 μL Buffer P1 and vortex.
3. Add 250 μL Buffer P2 and mix thoroughly by inverting the tube 4-6 times.
4. Add 350 μL Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.
5. Centrifuge for 10 min at 13,000g.
6. Apply the supernatant from step 5 to the QIAprep spin column by decanting or pippetting.
7. Centrifuge for 30-60 seconds. Discard the flow-through.
8. Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30-60 seconds. Discard the flow-through.
9. Wash QIAprep column by adding 0.75 ml Buffer PE and centrifuging for 30-60 seconds.
10. Discard the flow through, and centrifuge for an additional 1 minute to remove residual wash buffer.
11. To elute DNA, place the QIAprep column in a clean 1.5 ml microcentrifuge tube. Add 50 μL PCR water (if sample is to be sequenced) or 50 μL elution solution to the center of each QIAprep spin column, let stand for 1 minute, and centrifuge for 1minute.

Sigma Aldrich miniprep

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The following protocol is applicable to the [http://www.sigmaaldrich.com/life-science/molecular-biology/dna-and-rna-purification/plasmid-miniprep-kit.html GenElute™ Plasmid Miniprep Kit], using micro-centrifuge:

1. Add 1.5 mL of liquid culture to microcentrifuge tube and centrifuge at 13,000 rpm for 1 minute. Discard supernatant.
2. Resuspend pelleted bacterial cells in 200 μL resuspension solution. Vortex or pipette up and down to completely resuspend cells.
3. Lyse cells with 200 μL lysis buffer. Mix by gentle inversion- do not vortex.
4. Add 350 μL neutralization buffer to precipitate cell debris. Mix by gentle inversion.
5. Prep provided binding column by adding 500 μL column preparation solution and centrifuging at 12,000g for 1 minute.
6. Transfer the cell material from step 4 to the column and centrifuge at 12,000g for 1 minute. Discard flow through.
7. Add 500 μL wash solution 1. Centrifuge at 12,000g for 1 minute. Discard flow through.
8. Add 750 μL wash solution 2. Centrifuge at 12,000g for 1 minute. Discard flow through.
9. Transfer the column to a new collection tube.
10. Add 100 μL PCR water (if sample is to be sequenced) or 100 μL elution solution to the center of each column. Centrifuge at 12,000g for 1 minute. The DNA is now collected in the flow through liquid.

Genome prep

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We extract bacterial DNA to use for PCR and sequencing. The protocol used throughout our project used the [http://www.sigmaaldrich.com/life-science/molecular-biology/dna-and-rna-purification/bacterial-genomic-miniprep-kit.html GenElute™ Bacterial Genomic DNA Kit].