Team:Cornell/Protocol
From 2011.igem.org
(Difference between revisions)
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==Molecular Cloning Protocols== | ==Molecular Cloning Protocols== | ||
- | + | ==='''PCR Reaction'''=== | |
:::<u>Note</u>: Keep everything on ice and add all volumes in a PCR tube. | :::<u>Note</u>: Keep everything on ice and add all volumes in a PCR tube. | ||
::::37.5μL ddH2O | ::::37.5μL ddH2O | ||
Line 20: | Line 20: | ||
:::Based on primers, set an appropriate annealing temperature | :::Based on primers, set an appropriate annealing temperature | ||
---- | ---- | ||
- | + | ==='''Agarose Gel Electrophoresis'''=== | |
:::#Prepare a 1% weight-to-volume agarose gel and add SYBR dye or ethidium bromide to stain DNA | :::#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) | :::#Place the gel in the apparatus rig with the wells facing the negative end (black-colored) | ||
Line 28: | Line 28: | ||
:::#Run at 120V | :::#Run at 120V | ||
---- | ---- | ||
- | + | ==='''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 | :::#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) | ||
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:::#Centrifuge at 13,000rpm for 1 minute | :::#Centrifuge at 13,000rpm for 1 minute | ||
---- | ---- | ||
- | + | ==='''DNA Quantification using NanoDrop Spectrophotometry'''=== | |
:::#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 | ||
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:::#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 | ||
---- | ---- | ||
- | + | ==='''Digestion Reaction'''=== | |
:::<u>Note</u>: Keep everything on ice | :::<u>Note</u>: Keep everything on ice | ||
::::? µL ddH2O (? = whatever volume needed to bring the total volume up to 50µL) | ::::? µL ddH2O (? = whatever volume needed to bring the total volume up to 50µL) | ||
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:::*Incubate the digestion reaction tube in a 37°C water bath for 3 hours | :::*Incubate the digestion reaction tube in a 37°C water bath for 3 hours | ||
---- | ---- | ||
- | + | ==='''Dephosphorylation of 5' Ends of Vector Backbone'''=== | |
:::#Add 1µL of Calf Intestinal Alkaline Phosphatase (CIAP) to the digested vector backbone | :::#Add 1µL of Calf Intestinal Alkaline Phosphatase (CIAP) to the digested vector backbone | ||
:::#Incubate at 50°C for 5 minutes | :::#Incubate at 50°C for 5 minutes | ||
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:::#Proceed to PCR clean up the sample | :::#Proceed to PCR clean up the sample | ||
---- | ---- | ||
- | + | ==='''PCR Clean Up of DNA''' ''(Qiagen QIAquick PCR Purification Kit)''=== | |
:::#Add 5 volumes of Buffer PB to 1 volume of PCR sample | :::#Add 5 volumes of Buffer PB to 1 volume of PCR sample | ||
:::#*ex: Add 250µL Buffer PB to 50µL PCR sample | :::#*ex: Add 250µL Buffer PB to 50µL PCR sample | ||
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:::#Centrifuge at 13,000rpm for 1 minute | :::#Centrifuge at 13,000rpm for 1 minute | ||
---- | ---- | ||
- | + | ==='''Ligation Reaction'''=== | |
:::<u>Note</u>: Keep everything on ice | :::<u>Note</u>: Keep everything on ice | ||
::::50-100ng vector backbone | ::::50-100ng vector backbone | ||
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:::*Incubate in 16°C water bath overnight | :::*Incubate in 16°C water bath overnight | ||
---- | ---- | ||
- | + | ==='''Desalting of Ligation Reaction Product'''=== | |
:::#Fill a petri dish with nanopure water | :::#Fill a petri dish with nanopure water | ||
:::#Place the desalting membrane on the water surface with the shiny side facing up | :::#Place the desalting membrane on the water surface with the shiny side facing up | ||
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:::#Wait 15 minutes | :::#Wait 15 minutes | ||
---- | ---- | ||
- | + | ==='''Transformation via Electroporation'''=== | |
:::#During the 15 minute wait of desalting, thaw electrocompetent bacterial cells on ice and cool the electroporation cuvette on ice | :::#During the 15 minute wait of desalting, thaw electrocompetent bacterial cells on ice and cool the electroporation cuvette on ice | ||
:::#Pipet up the desalted ligation mixture and add to thawed bacteria | :::#Pipet up the desalted ligation mixture and add to thawed bacteria | ||
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:::#Incubate the plate overnight at 37°C | :::#Incubate the plate overnight at 37°C | ||
---- | ---- | ||
- | + | ==='''PCR Deletion Reaction'''=== | |
:::<u>Note</u>: Keep everything on ice and add all volumes in a PCR tube. | :::<u>Note</u>: Keep everything on ice and add all volumes in a PCR tube. | ||
::::? µL ddH2O (? = whatever volume needed to bring the total volume up to 50µL) | ::::? µL ddH2O (? = whatever volume needed to bring the total volume up to 50µL) | ||
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:::*Volumes of diluted primer based on calculations for our ng/uL concentrations | :::*Volumes of diluted primer based on calculations for our ng/uL concentrations | ||
---- | ---- | ||
- | + | ==='''PCR Deletion Thermocycler Protocol'''=== | |
::::95°C for 2min | ::::95°C for 2min | ||
::::95°C for 30sec (18 times) | ::::95°C for 30sec (18 times) | ||
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:::*1min/kb corresponds to: 3.20min (RFP), 3.50min (VioA), 5.40min (VioB), 3.00min (VioE) | :::*1min/kb corresponds to: 3.20min (RFP), 3.50min (VioA), 5.40min (VioB), 3.00min (VioE) | ||
---- | ---- | ||
- | + | ==='''Preparing a DNA Sample for Sequencing'''=== | |
:::- 1µL primer (8 pmol -- ex: 8µL 100mM stock primer + 92µL ddH2O) | :::- 1µL primer (8 pmol -- ex: 8µL 100mM stock primer + 92µL ddH2O) | ||
:::- at least 1µg DNA | :::- at least 1µg DNA |
Revision as of 20:18, 18 September 2011
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.
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
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
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
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
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
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
Desalting of Ligation Reaction Product
- Fill a petri dish with nanopure water
- Place the desalting membrane on the water surface with the shiny side facing up
- Add 7µL ligation reaction product onto the membrane
- Wait 15 minutes
Transformation via Electroporation
- During the 15 minute wait of desalting, thaw electrocompetent bacterial cells on ice and cool the electroporation cuvette on ice
- Pipet up the desalted ligation mixture and add to thawed bacteria
- Transfer bacterial cell mixture to cuvette and keep on ice
- Pulse the cuvette using the electroporator at "E. coli: 1mm and 1.8kV" settings
- Add 900µL SOB to the cuvette, pipet mix, and transfer entire volume to the original Eppendorf containing the frozen bacteria
- Shake the transformation product at 37°C for 1.5 hours
- Plate the cells on an agar plate treated with the appropriate antibiotic
- Incubate the plate overnight at 37°C
PCR Deletion Reaction
- Note: Keep everything on ice and add all volumes in a PCR tube.
- ? µL ddH2O (? = whatever volume needed to bring the total volume up to 50µL)
- 5.0μL 10x PfuUltra buffer
- 1.0μL dNTPs
- ? uL forward primer = 125ng fwd primer ÷ fwd primer concentration (ng/µL)
- ? uL reverse primer = 125ng rvs primer ÷ rvs primer concentration (ng/µL)
- ? µL dsDNA= 20ng insert ÷ insert concentration (ng/µL)
- 1.0μL PfuUltra
- 50.0μL Total
- Volumes of diluted primer based on calculations for our ng/uL concentrations
- Note: Keep everything on ice and add all volumes in a PCR tube.
PCR Deletion Thermocycler Protocol
- 95°C for 2min
- 95°C for 30sec (18 times)
- 55°C for 30sec
- 72°C for 1 min/kb
- 1min/kb corresponds to: 3.20min (RFP), 3.50min (VioA), 5.40min (VioB), 3.00min (VioE)
Preparing a DNA Sample for Sequencing
- - 1µL primer (8 pmol -- ex: 8µL 100mM stock primer + 92µL ddH2O)
- - at least 1µg DNA
- - fill up to 18µL total volume with ddH2O