Team:Nevada/Protocols

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Bacterial Glycerol Stocks

1. Put 0.5mL bacterial culture in a sterile eppendorf tube.
2. Add 0.5mL of sterile 80% (v/v) glycerol solution
3. Freeze in liquid nitrogen and add to -80oC freezer
4. To recover, scrape frozen surface of culture with sterile inoculating needle, and then streak onto LB agar plate containing appropriate antibiotics, or inoculate liquid culture containing appropriate antibiotics.

DNA Quantification using NanoDrop Spectrophotometry

1. Select Nucleic Acids measurement
2. Initialize the NanoDrop spectrophotometer with 2µL of autoclaved H2O and wipe off
3. Blank (calibrate) the NanoDrop spectrophotometer with 2µL of the same elution buffer used during DNA purification and wipe off
4. Measure 1.5µL of DNA sample and record the concentration in ng/µL

Gel Purification of DNA (Qiagen QIAquick Gel Extraction Kit)

1. Cut out the DNA fragment from the agarose gel with a razor blade, while minimizing the size of the gel slice
2. Weigh the gel slice and add 3 volumes of Buffer QG to every 1 volume of gel (100mg = 100µL)
3. Dissolve the gel slice using a 60°C heat block
4. Apply the dissolved gel to the QIAquick column and centrifuge at 13,000rpm for 1 minute
5. Discard the flow-through and repeat Step 4 until all sample has passed through the column
6. Add 500µL of Buffer QG to the QIAquick column and centrifuge at 13,000rpm for 1 minute
7. Wash the column with 750µL of Buffer PE and centrifuge at 13,000rpm for 1 minute
8. Discard the flow-through and centrifuge at 13,000rpm for 1 minute to remove residual EtOH
9. Transfer the QIAquick column to a new Eppendorf
10. Add 35µL elution buffer to the center of the column and wait at least 2 minutes
11. Centrifuge at 13,000rpm for 1 minute

PCR Purification of DNA

1. Add 5 volumes of Buffer DB to 1 volume of PCR sample
   • ex: Add 250µL Buffer DB to 50µL PCR sample
2. Apply this mixture to a QIAquick column and centrifuge at 13,000rpm for 1 minute
3. Discard flow-through and repeat Step 2 until all sample has passed through the column
4. Wash column with 750µL Buffer PE and centrifuge at 13,000rpm for 1 minute
5. Discard flow-through and centrifuge at 13,000rpm for 1 minute to remove residual EtOH
6. Transfer QIAquick column to new Eppendorf
7. Apply 50µL elution buffer to center of the column and wait at least 2 minutes
8. Centrifuge at 13,000rpm for 1 minute

Phusion PCR

• Thermocycling conditions:
  

1. Initial Denaturation: 98°C for 30 seconds
   
2. 25-35 cycles:
   

• 98°C for 10 seconds
• 55°C, 60°C, 65°C for 15 seconds
• 72°C for 15 seconds

1. Final Extension: 72°C for 5 minutes
   

Qiagen Miniprep kit

www.qiagen.com/hb/qiaprepminiprep