Team:Cornell/Protocol
From 2011.igem.org
Results |
Protocol |
Notebook |
Parts Submitted
Protocols
- Below, please find the steps that we followed to carry out molecular cloning, create recombinant DNA parts, and construct microfluidic channels.
- Below, please find the steps that we followed to carry out molecular cloning, create recombinant DNA parts, and construct microfluidic channels.
Molecular Cloning Protocols
- PCR Reaction
- Note: Keep everything on ice and add all volumes in a PCR tube.
- 37.5μL ddH2O
- 5.0μL 10x buffer
- 2.5μL dNTPs
- 1.0μL MgSO4
- 1.0μL forward primer
- 1.0μL reverse primer
- 1.0μL template
- 1.0μL DNA polymerase
- 50.0μL Total
- Based on primers, set an appropriate annealing temperature
- Note: Keep everything on ice and add all volumes in a PCR tube.
- PCR Reaction
- Agarose Gel Electrophoresis
- Prepare a 1% weight-to-volume agarose gel and add SYBR dye or ethidium bromide to stain DNA
- Place the gel in the apparatus rig with the wells facing the negative end (black-colored)
- Fill the rig with 1x TBE buffer
- Load 2µL of 1kb ladder
- Add 2µL of 6x loading dye to each PCR reaction tube. Load 20µL in wells
- Run at 120V
- Agarose Gel Electrophoresis
- 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
- Gel Purification of DNA (Qiagen QIAquick Gel Extraction Kit)
- 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
- DNA Quantification using NanoDrop Spectrophotometry
- Digestion Reaction
- Note: Keep everything on ice
- ? µL ddH2O (? = whatever volume needed to bring the total volume up to 50µL)
- 5µL 10x NEBuffer
- ? µL DNA sample (? = whatever volume corresponds with 1µg)
- 0.5µL 100x BSA
- 1µL first restriction enzyme
- 1µL second restriction enzyme
- 50µL Total
- Note: Consult www.neb.com to determine the buffer compatibility of the restriction enzymes used
- Incubate the digestion reaction tube in a 37°C water bath for 3 hours
- Note: Keep everything on ice
- Digestion Reaction
- Dephosphorylation of 5' Ends of Vector Backbone
- Add 1µL of Calf Intestinal Alkaline Phosphatase (CIAP) to the digested vector backbone
- Incubate at 50°C for 5 minutes
- Inactivate CIAP by heating at 85°C for 15 minutes
- Proceed to PCR clean up the sample
- Dephosphorylation of 5' Ends of Vector Backbone
- PCR Clean Up of DNA (Qiagen QIAquick PCR Purification Kit)
- Add 5 volumes of Buffer PB to 1 volume of PCR sample
- ex: Add 250µL Buffer PB 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 PB to 1 volume of PCR sample
- PCR Clean Up of DNA (Qiagen QIAquick PCR Purification Kit)
- Ligation Reaction
- Note: Keep everything on ice
- 50-100ng vector backbone
- 3:1 molar ratio of insert:vector
- - X ng insert = (3 * Y ng vector * A bp insert) ÷ (B bp vector)
- - ? µL insert = X ng insert ÷ insert concentration (ng/µL)
- ? µL autoclaved H2O (? = whatever volume needed to bring the total volume to 20µL)
- 2µL 10x T4 DNA ligase buffer
- 1µL T4 DNA ligase
- 20µL Total
- Incubate in 16°C water bath overnight
- Note: Keep everything on ice
- Ligation Reaction