Team:Nevada/Protocols
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==Phusion PCR== | ==Phusion PCR== | ||
- | Thermocycling conditions:<br/> | + | ::*Thermocycling conditions:<br/> |
- | Initial Denaturation: 98°C for 30 seconds <br/> | + | :::#Initial Denaturation: 98°C for 30 seconds <br/> |
- | 25-35 cycles: <br/> | + | :::#25-35 cycles: <br/> |
- | * 98°C for 10 seconds | + | ::::* 98°C for 10 seconds |
- | * 55°C, 60°C, 65°C for 15 seconds | + | ::::* 55°C, 60°C, 65°C for 15 seconds |
- | * 72°C for 15 seconds | + | ::::* 72°C for 15 seconds |
- | Final Extension: 72°C for 5 minutes<br/> | + | :::#Final Extension: 72°C for 5 minutes<br/> |
==Qiagen Miniprep kit== | ==Qiagen Miniprep kit== | ||
www.qiagen.com/hb/qiaprepminiprep<br/> | www.qiagen.com/hb/qiaprepminiprep<br/> |
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Contents |
Gel Purification of DNA (Qiagen QIAquick Gel Extraction Kit)
- Cut out the DNA fragment from the agarose gel with a razor blade, while minimizing the size of the gel slice
- Weigh the gel slice and add 3 volumes of Buffer QG to every 1 volume of gel (100mg = 100µL)
- Dissolve the gel slice using a 60°C heat block
- Apply the dissolved gel to the QIAquick column and centrifuge at 13,000rpm for 1 minute
- Discard the flow-through and repeat Step 4 until all sample has passed through the column
- Add 500µL of Buffer QG to the QIAquick column and centrifuge at 13,000rpm for 1 minute
- Wash the column with 750µL of Buffer PE and centrifuge at 13,000rpm for 1 minute
- Discard the flow-through and centrifuge at 13,000rpm for 1 minute to remove residual EtOH
- Transfer the QIAquick column to a new Eppendorf
- Add 35µL elution buffer to the center of the column and wait at least 2 minutes
- Centrifuge at 13,000rpm for 1 minute
DNA Quantification using NanoDrop Spectrophotometry
- Select Nucleic Acids measurement
- Initialize the NanoDrop spectrophotometer with 2µL of autoclaved H2O and wipe off
- Blank (calibrate) the NanoDrop spectrophotometer with 2µL of the same elution buffer used during DNA purification and wipe off
- Measure 1.5µL of DNA sample and record the concentration in ng/µL
PCR Purification of DNA
- Add 5 volumes of Buffer DB to 1 volume of PCR sample
- ex: Add 250µL Buffer DB to 50µL PCR sample
- Apply this mixture to a QIAquick column and centrifuge at 13,000rpm for 1 minute
- Discard flow-through and repeat Step 2 until all sample has passed through the column
- Wash column with 750µL Buffer PE and centrifuge at 13,000rpm for 1 minute
- Discard flow-through and centrifuge at 13,000rpm for 1 minute to remove residual EtOH
- Transfer QIAquick column to new Eppendorf
- Apply 50µL elution buffer to center of the column and wait at least 2 minutes
- Centrifuge at 13,000rpm for 1 minute
- Add 5 volumes of Buffer DB to 1 volume of PCR sample
Phusion PCR
- Thermocycling conditions:
- Initial Denaturation: 98°C for 30 seconds
- 25-35 cycles:
- 98°C for 10 seconds
- 55°C, 60°C, 65°C for 15 seconds
- 72°C for 15 seconds
- Final Extension: 72°C for 5 minutes
- Initial Denaturation: 98°C for 30 seconds
- Thermocycling conditions: