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From 2011.igem.org

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

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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

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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

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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

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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

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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

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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

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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

Microfluidics Protocols