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Revision as of 00:03, 20 September 2011

Protocols

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

Agarose Gel Electrophoresis

1. Prepare a 1% weight-to-volume agarose gel and add SYBR dye or ethidium bromide to stain DNA
2. Place the gel in the apparatus rig with the wells facing the negative end (black-colored)
3. Fill the rig with 1x TBE buffer
4. Load 2µL of 1kb ladder
5. Add 2µL of 6x loading dye to each PCR reaction tube. Load 20µL in wells
6. Run at 120V

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

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

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

Dephosphorylation of 5' Ends of Vector Backbone

1. Add 1µL of Calf Intestinal Alkaline Phosphatase (CIAP) to the digested vector backbone
2. Incubate at 50°C for 5 minutes
3. Inactivate CIAP by heating at 85°C for 15 minutes
4. Proceed to PCR clean up the sample

PCR Clean Up of DNA (Qiagen QIAquick PCR Purification Kit)

1. Add 5 volumes of Buffer PB to 1 volume of PCR sample
   • ex: Add 250µL Buffer PB 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

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

Desalting of Ligation Reaction Product

1. Fill a petri dish with nanopure water
2. Place the desalting membrane on the water surface with the shiny side facing up
3. Add 7µL ligation reaction product onto the membrane
4. Wait 15 minutes

Transformation via Electroporation

1. During the 15 minute wait of desalting, thaw electrocompetent bacterial cells on ice and cool the electroporation cuvette on ice
2. Pipet up the desalted ligation mixture and add to thawed bacteria
3. Transfer bacterial cell mixture to cuvette and keep on ice
4. Pulse the cuvette using the electroporator at "E. coli: 1mm and 1.8kV" settings
5. Add 900µL SOB to the cuvette, pipet mix, and transfer entire volume to the original Eppendorf containing the frozen bacteria
6. Shake the transformation product at 37°C for 1.5 hours
7. Plate the cells on an agar plate treated with the appropriate antibiotic
8. Incubate the plate overnight at 37°C

PCR Deletion (Site-Directed Mutagenesis) 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 high-fidelity DNA polymerase

50.0μL Total

• Volumes of diluted primer based on calculations for our ng/µL concentrations

PCR Deletion (Site-Directed Mutagenesis) 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

Microfluidics Protocols