Team:Nevada/Protocols
From 2013.igem.org
(Difference between revisions)
Line 7: | Line 7: | ||
:::#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. | :::#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 | + | ==DNA Quantification using NanoDrop== |
:::#Select ''Nucleic Acids'' measurement | :::#Select ''Nucleic Acids'' measurement | ||
:::#Initialize the NanoDrop spectrophotometer with 2µL of autoclaved H2O and wipe off | :::#Initialize the NanoDrop spectrophotometer with 2µL of autoclaved H2O and wipe off | ||
Line 13: | Line 13: | ||
:::#Measure 1.5µL of DNA sample and record the concentration in ng/µL | :::#Measure 1.5µL of DNA sample and record the concentration in ng/µL | ||
- | ==Gel Purification of DNA | + | ==Gel Purification of DNA== |
:::#Cut out the DNA fragment from the agarose gel with a razor blade, while minimizing the size of the gel slice | :::#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) | :::#Weigh the gel slice and add 3 volumes of Buffer QG to every 1 volume of gel (100mg = 100µL) |
Revision as of 06:31, 27 September 2013
Bacterial Glycerol Stocks
- Put 0.5mL bacterial culture in a sterile eppendorf tube.
- Add 0.5mL of sterile 80% (v/v) glycerol solution
- Freeze in liquid nitrogen and add to -80oC freezer
- 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
- 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
Gel Purification of DNA
- 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
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:
Qiagen Miniprep kit
- www.qiagen.com/hb/qiaprepminiprep
- www.qiagen.com/hb/qiaprepminiprep