Team:Cooper Union/Experiments

Cooper Union 2015 iGEM



1 - Bacteria Media w/ agar (solid media)
2 - Dynabead Prep
3 - Bacterial Transformation
4 - Colony PCR of pSB1C3 with TdT Mutant Inserts
5 - Dynabead Cleanamp
6 - Double Digest of PET, PSB, TdT variants
7 - Immobilizing Disulfide Modified Oligos to Mercaptosilane Treated Glass Slides
8 - RNA Ligase Reaction
9 - Gel Extraction
10 - Miniprep
11 - PCR Purification
12 - TdT Nucleotide Addition
13 - Non-Radioactive phosphorlyation with T4 PNK

Experiments & Protocols



1- Bacteria Media w/ agar (solid media)

Materials
  • 500mL bottle
  • Deionized Water
  • Agar
  • LB Broth
  • Chloramphenicol
  • Yeast Extract
  • Bacto Peptone
    • Procedure
    1. Add 250mL of deionized water to the 500mL bottle.
    2. Weigh out 3.625g of agar using the weighing boat and add it to the 250mL of water.
    3. Weigh out 5.0g of LB Broth using the weighing boat and add it to the 250mL of water.
    4. Screw lid on bottle loosely.
    5. Place autoclave tape on the lid of the bottle.
    6. Place tray with approximately 1cm of water in height into the autoclave.
    7. Place 500mL bottle into autoclave with "liquid" selected and press "start".
    8. After an hour, remove bottle from autoclave. Let bottle sit for approximately 30 minutes.
    9. Add 370uL of chloramphenicol to the 250mL solution.
    10. Pour solution into petri dishes, covering the bottom of the dish.
    11. Place two stripes on each dish.
    12. Let dishes sit overnight.

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    2 - Dynabead Prep

    Materials
  • B&W wash Buffer (2X and 1X)
  • Dynabeads
  • DNA solution
  • Magnets
  • PCR Tubes
    • Procedure
    1. Pipette 50 microL of dynabeads in PCR tubes (3 of them)
    2. Wash beads by pipetting in 200 microL of 1X B&W buffer
    3. Vortex PCR tubes for about 30 seconds
    4. Place a magnet at the bottom of the tube until a solid pellet of dynabeads forms at the bottom
    5. Pipette out the supernatant leaving the pellet intact at the bottom
    6. Repeat steps 2-5 two times (performed a total 3 times)
    7. To each tube add 100 microL 2X buffer, 5 microL DNA (100 microM), and 95 microL of water
    8. Incubate on stirring apparatus/agitator at room temperature for 15 minutes
    9. Wash the beads again by repeating steps 2-5 three times
    10. Add 105 microL 1X buffer to the tubes to resuspend the beads
    11. Freeze PCR tubes at -20 degrees Celsius until they are needed

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    3 - Bacterial Transformation

  • For C2987H: Thaw a tube of NEB 5-alpha Competent E. coli cells on ice for 10 minutes.
  • For C2987I: Thaw a tube of NEB 5-alpha Competent E. coli cells on ice until the last ice crystals disappear. Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice.
  • Add 1-5 µl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
  • Place the mixture on ice for 30 minutes. Do not mix.
  • Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
  • Place on ice for 5 minutes. Do not mix.
  • Pipette 950 µl of room temperature SOC into the mixture.
  • Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
  • Warm selection plates to 37°C.
  • Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.
  • Spread 50-100 µl of each dilution onto a selection plate and incubate overnight at 37°C.
  • Alternatively, incubate at 30°C for 24-36 hours or 25°C for 48 hours.

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    4 - Colony PCR of pSB1C3 with TdT Mutant Inserts

    Materials
  • VR Primer
  • VF2 Primer
  • pSB1C3 colonies
  • ddH20
  • 10 mm dNTPs
  • Taq Polymerase
  • 10X Standard Taq Polymerase Buffer
  • 6X Loading Dye
  • DNA Ladder
  • 1% Agarose Gel
    • Procedure
    1. Use a pipette tip to scrape off a colony and place the colony in 200 microL of ddH20
    2. Take 21 microL of bacteria water and add it to a PCR tube
    3. Add half a microliter of primer VR and another half microliter of primer VF2 to PCR tube
    4. Add half a microliter of 10 mm dNTPs, 0.125 microliters of Taq Polymerase, and the appropriate amount of Standard Taq Polymerase Buffer (Usually 2.5 for a 25 microliter reaction)
    5. Run in PCR machine with desired thermal cycling
    6. Prepare a 1% agarose gel if one is not available
    7. Prepare sample for gel electrophoresis using a proper proportion of 6X loading dye to sample
    8. Load DNA ladder and sample into gel and run gel
    9. Analyze gel using U.V. light and determine the approximate length of the amplified sequence

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    5 - Dynabead Cleanamp

    Materials
  • 10x TdT buffer
  • 2.5 mM CoCl2
  • Oligo
  • 50 mM CleanAmp dATP
  • 100 mM dATP
  • TdT
  • dH2O
    • Procedure
    1. Preheat water baths to 37 celsius and 95 celsius.
    2. Mix: - 5 µl 10x TdT buffer, - 5 µl 2.5 mM CoCl2, - 1.3 µl of 43 mer oligo ( 1 µg at 813 ng/µl), - 0.4 µl 50 mM CleanAmp dATP, - 1 µl TdT ( 20 units at 20000 units/mL), - dH2O to final volume of 50 mL.
    3. Split into two separate equal volume aliquots.
    4. Incubate at 37 celsius for 30 minutes.
    5. Incubate 1 aliquot at 95 celsius for 15 minutes.
    6. For the non-CleanAmp dATP, replace CleanAmp dATP with 0.2 µl 100 mM dATP.
    7. For the negative control, remove the volume of TdT.
    8. Store at -20 celsius.

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    6 - Double Digest of PET, PSB, TdT variants

    Materials
  • PET28
  • PSB1C3
  • Nuclease-free water
  • EcoR1-HF
  • Pst
  • XbaI
  • BamhI
  • TdT (TdT del1-27, del26-143, & GIP subAAA 213-215)
  • NEBuffer 3.1
  • NEBuffer 2.1
    • Procedure
    1. Digest of PET28 and PSB1C3
      1. Mix together these materials in this order for PET28 tube:
        1. 3.1 buffer---5uL
        2. Water---32.2uL
        3. XbaI---1uL
        4. BamhI---1uL
        5. PET28---10.8uL
      2. Mix together these materials in this order for PSB1C3 tube:
          3. ​
        1. 2.1 buffer---5uL
        2. Water---31.2uL
        3. EcoRI---1uL
        4. PstI---1uL
        5. PSB1C3---11.8uL
      3. Incubate both tubes at 37˚C for 1 hour
    2. Digestion of TdT sequences
      1. Take three tubes and mix together these ingredients for each:
        1. 3.1 buffer---1.5uL
        2. Water---2.5uL
        3. XbaI---0.5uL
        4. BamhI---0.5uL
      2. Place each TdT variant in each tube
      3. Take three more tubes and mix together these ingredients for each:
        1. 2.1 buffer---1.5uL
        2. Water---2.5uL
        3. EcoRI---0.5uL
          4. PstI---0.5uL
      4. Place each TdT variant in each tube
      5. Incubate both tubes at 37˚C for 1 hour
      6. Purify TdT DNA
        1. tubes containing EcoRI and PstI are purified by incubating at 80˚C for 20 minutes
        2. tubes containing XbaI and PstI are purified using life technologies "Procedure for Purifying PCR Products" (in orange folder)

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    7 - Immobilizing Disulfide Modified Oligos to Mercaptosilane Treated Glass Slides

    Materials
  • Untreated Glass Slides or Glass Coverslips
  • Disulfide Modified Oligonucleotides (Ordered from IDT with the C6 S-S 5’ Thiol Modifier)
  • 500 mM Sodium Bicarbonate Buffer (pH 9.0)
  • Anhydrous Ethanol
  • 25% Ammonium Hydroxide
  • 1% 3-mercaptopropyl trimethoxysilane, 95% Ethanol, 16 mM acetic acid (pH 4.5)
  • 95% Ethanol, 16 mM acetic acid (pH 4.5)
  • 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% Tween 20
  • Running RO water or ddH20
  • Vacuum Oven
  • Vacuum Pump
  • Fume Hood
  • Petri dishes
  • Parafilm
    1. Place untreated glass slides in a clean Petri dish
    2. Submerge glass slides in 25% Ammonium Hydroxide overnight
    3. Rinse the same glass slides under running RO water for 10 minutes
    4. Rinse the slides with Anhydrous Ethanol
    5. Immerse the slides in 1% 3-mercaptopropyl trimethoxysilane, 95% Ethanol, 16 mM acetic acid (pH 4.5) for 30 - 45 minutes
    6. Rinse the same slides with 95% Ethanol, 16 mM acetic acid (pH 4.5)
    7. Cure the glass slides in a vacuum oven at 150C for 2 hours
    8. While slides are curing, suspend 1 - 40 uM of disulfide modified DNA in 150 uL of of 500 mM Sodium Bicarbonate Buffer (pH 9.0)
    9. Remove cured slides from vacuum oven, and array disulfide modified DNA suspended in 500 mM Sodium Bicarbonate Buffer (pH 9.0) onto slides. Various arrays, ranging from 5 x 5 lattices using 2 uL dots to 1 large 50 uL dot were used
    10. Place the slides in a moist environment, such as on a rack in a 30C water bath, overnight
    11. Clean the slides using three washes of 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% Tween 20
    12. Store the slides in 4C temperature

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    8 - RNA Ligase Reaction

    Materials
  • 1 ug Single Stranded DNA with 5’ Phosphate (X mer) [Retrieved as an IDT Custom Oligo]
  • 1 ug Single Stranded DNA (Y mer) [Retrieved as an IDT Custom Oligo or can be any primer]
  • T4 RNA Ligase 1
  • T4 RNA Ligase 1 Reaction Buffer
  • PEG 8000
  • ATP
  • 10 mM Tris-HCl pH 8.0, 2.5 mM EDTA
    • Procedure
    1. Make a 20 uL reaction that contains the following:
      • 1 ug of ssDNA with 5’ Phosphate
      • 1 ug of ssDNA (Any primer or oligo)
      • 25% PEG 8000
      • 1 mM ATP
      • 1X T4 RNA Ligase 1 Reaction Buffer
      • uL of T4 RNA Ligase 1
    2. Incubate the mixture overnight for 16-18 hours at room temperature
    3. Terminate the reaction by adding 40 uL of 10 mM Tris-HCl pH 8.0, 2.5 mM EDTA

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    9 - Gel Extraction

    Materials
  • DNA sample ran through agarose gel
  • Sharp edge, like scalpel or gel cutter
  • Qiagen Buffers QG, PE, EB
  • Isopropanol
    • Procedure
    1. Prewarm waterbath to 50°C
    2. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
    3. Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg ~ 100 µl)
    4. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). To help dissolve gel, mix by vortexing the tube every 2–3 min during the incubation.
    5. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose).
    6. Add 1 gel volume of isopropanol to the sample and mix.
    7. Place a QIAquick spin column in a provided 2 ml collection tube.
    8. To bind DNA, apply the sample to the QIAquick column, and centrifuge for 1 min.
    9. Discard flow-through and place QIAquick column back in the same collection tube. (Optional): Add 0.5 ml of Buffer QG to QIAquick column and centrifuge for 1 min.
    10. To wash, add 0.75 ml of Buffer PE to QIAquick column and centrifuge for 1 min. Discard the flow-through and centrifuge the QIAquick column for an additional 1 min at ≥10,000 x g (~13,000 rpm).
    11. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
    12. To elute DNA, add 50 µl of Buffer EB (10 mM Tris·Cl, pH 8.5) or H2O to the center of the QIAquick membrane and centrifuge the column for 1 min at maximum speed.

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    10 - Miniprep

    Materials
  • 5mL of overnight bacteria culture
  • Qiagen Buffers P1, P2, N3, PE, EB
    • Procedure
    1. Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a microcentrifuge tube.
    2. Add 250 µl Buffer P2 and gently invert the tube 4–6 times to mix
    3. Add 350 µl Buffer N3 and invert the tube immediately but gently 4–6 times.
    4. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
    5. Apply the supernatants from step 4 to the QIAprep spin column by decanting or pipetting.
    6. Centrifuge for 30–60 s. Discard the flow-through. (Optional): Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard the flow-through.
    7. Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s.
    8. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.
    9. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 µl
    10. Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.

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    11 - PCR Purification

    Materials
  • PCR Reaction to be purified
  • Qiagen Buffers PB, PE, EB
    • Procedure
    1. Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix.
    2. If pH indicator has been added to Buffer PB, check that the color of the mixture is yellow.
    3. Place a QIAquick spin column in a provided 2 ml collection tube.
    4. To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 s.
    5. Discard flow-through. Place the QIAquick column back into the same tube.
    6. To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30–60 s.
    7. Discard flow-through and place the QIAquick column back in the same tube.
    8. Centrifuge the column for an additional 1 min.
    9. Place QIAquick column in a clean 1.5 ml microcentrifuge tube.
    10. To elute DNA, add 50 µl Buffer EB (10 mM Tris·Cl, pH 8.5) or water (pH 7.0–8.5) to the center of the QIAquick membrane and centrifuge the column for 1 min.

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    12 - TdT Nucleotide Addition

    Materials
  • DNA source - commercially made oligo's
  • Terminal Transferase
  • CleanAmp dNTP's
    • Procedure
  • Repeat for each nucleotide
    1. Pre-heat water baths to 37°C and 95°C
    2. Mix:
    1. 5.0 μL 10X TdT Buffer
    2. 5.0 μL 2.5 mM CoCl2 solution
    3. 1μg 43 mer oligo (1.3μL of 813 ng/μL)
    4. 0.4 μL 50mM CleanAmp dATP
    5. 20 Units TdT (1 μL of 20,000 U/μL)
    6. dH2O to final volume of 50 μL
    7. Incubate at 37°C for 30 minutes.
    8. Prepare 2 equal volume aliquots of the mixture (25 μL each).
    9. Incubate 1 aliquot at 95°C for 15 minutes.
    10. Add additional 10 U TdT (0.5 μL of 20,000 U/μL) to both aliqouts.
    11. Cool 95°C water bath to 70°C.
    12. Incubate at 37°C for 60 minutes.
    13. Heat inactivate at 70°C for 10 minutes.
    14. Store at -20°C.
  • Controls
    1. Negative (No TdT)
      1. Pre-heat water baths to 37°C and 70°C
      2. Mix:
      1. 5.0 μL 10X TdT Buffer
      2. 5.0 μL 2.5 mM CoCl2 solution
      3. 500 ng 24-mer oligo (0.9 μL of 568.8 ng/μL)
      4. 1.0 μL 10mM dATP
      5. dH2O to final volume of 50 μL
      6. Incubate at 37°C for 60 minutes
      7. Heat inactivate at 70°C for 10 minutes.
      8. Store at -20°C.
    2. Five Minute Incubation Time
      1. Pre-heat water baths to 37°C and 70°C
      2. Mix:
      1. 5.0 μL 10X TdT Buffer
      2. 5.0 μL 2.5 mM CoCl2 solution
      3. 500 ng 24-mer oligo (0.9 μL of 568.8 ng/μL)
      4. 1.0 μL 10mM dATP
      5. 10 Units TdT (0.5 μL of 20,000 U/μL)
      6. dH2O to final volume of 50 μL
      7. Incubate at 37°C for 5 minutes
      8. Heat inactivate at 70°C for 10 minutes.
      9. Store at -20°C.
      Sixty Minute Incubation Time
      1. Pre-heat water baths to 37°C and 70°C
      2. Mix:
      1. 5.0 μL 10X TdT Buffer
      2. 5.0 μL 2.5 mM CoCl2 solution
      3. 500 ng 24-mer oligo (0.9 μL of 568.8 ng/μL)
      4. 1.0 μL 10mM dATP
      5. 10 Units TdT (0.5 μL of 20,000 U/μL)
      6. dH2O to final volume of 50 μL
      7. Incubate at 37°C for 60 minutes
      8. Heat inactivate at 70°C for 10 minutes.
      9. Store at -20°C.

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    13- Non-Radioactive phosphorlyation with T4 PNK

    Procedure
    1. Mix the following:
    • up to 300 pmol of single stranded oligo (43mer)
    • 5 microliter of T410X PNK Buffer
    • 5 microliter of 10 mM ATP
    • 1 microliter of PNK
    • ddH20 upto a total volume of 50 uL

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    References

    3 : Bacterial Transformation - https://www.neb.com/protocols/1/01/01/high-efficiency-transformation-protocol-c2987
    4 : Colony PCR of pSB1C3 with TdT Mutant Inserts - https://www.neb.com/protocols/1/01/01/taq-dna-polymerase-with-standard-taq-buffer-m0273
    7 : Immobilizing Disulfide Modified Oligos to Mercaptosilane Treated Glass Slides - http://xobi.net/Lib/AnalBio99v266p23.pdf
    8 : RNA Ligase Reaction - https://www.neb.com/protocols/1/01/01/ligation-of-an-oligo-to-single-stranded-dna-cdna-using-t4-rna-ligase-1
    9 : Gel Extraction - http://www.aun.edu.eg/molecular_biology/gene&protein/Gel%20extraction-Qiagen.pdf
    10 : Miniprep - http://stanxterm.aecom.yu.edu/wiki/data/Product_manuals_attach/qiagenmini.pdf
    11 : PCR Purification - https://static.igem.org/mediawiki/2012/a/a3/QIAquick_PCR-purification.pdf
    12 : TdT Nucleotide Addition - http://www.neb.com/protocols/1/01/01/non-radioactive-phosphorylation-with-t4-pnk-or-pnk3-phosphatase-minus