Team:Cooper Union/Protocols

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Cooper Union 2014 iGEM



Common Laboratory Protocols






Bacterial Transformation


Materials
plasmid DNA (up to 100ng, no more than 5μL)
Competent bacteria aliquot (stored -80°C)
SOC (stored 4°C)
Procedure
  1. Prewarm 2 water baths to 37°C and 42°C
  2. Aliquot 100μL SOC into microcentrifuge tube. Prewarm in 37°C.
  3. Prewarm LB plate + resistance to 37°C.
  4. Add DNA to compontent cells. Incubate on ice 15-20 min.
  5. Heat shock by quickly transferring tubes from ice to 42°C water bath for EXACTLY 45 seconds.
  6. Recuperate for 2 minutes on ice.
  7. Transfer prewarmed SOC to bacteria. Incubate 30 min 37°C.
  8. Transfer all the solution to LB plate. Spread and wait 15 min on benchtop.
  9. Invert and incubate 37°C overnight.


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Colony PCR



Gel Extraction

Qiagen protocol generally used
  1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
  2. Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of
  3. gel (100 mg ~ 100 µl)
  4. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). To help
  5. dissolve gel, mix by vortexing the tube every 2–3 min during the incubation.
  6. After the gel slice has dissolved completely, check that the color of the mixture is
  7. yellow (similar to Buffer QG without dissolved agarose).
  8. Add 1 gel volume of isopropanol to the sample and mix.
  9. Place a QIAquick spin column in a provided 2 ml collection tube.
  10. To bind DNA, apply the sample to the QIAquick column, and centrifuge for 1 min.
  11. Discard flow-through and place QIAquick column back in the same collection tube.
  12. Optional): Add 0.5 ml of Buffer QG to QIAquick column and centrifuge for 1 min.
  13. To wash, add 0.75 ml of Buffer PE to QIAquick column and centrifuge for 1 min.
  14. Discard the flow-through and centrifuge the QIAquick column for an additional 1 min
  15. at ≥10,000 x g (~13,000 rpm).
  16. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
  17. 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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Miniprep



PCR Purification



PCR Reaction



Restriction Enzyme Digest


Materials
10x Buffer (Compatible to the enzyme)
Enzyme
BSA - diluted to 10x
ddH2O
DNA - up to 1μg per reaction.

Procedure Verify all times and temperatures for specific enzyme prior to use. This is general protocol.
  1. Prewarm 2 water baths to 37°C and 70°C
  2. Thaw buffers, diluted BSA. Keep all reagents on ice
  3. Set up reactions based on table below.
    ReagentVolumeExample
    Reaction Buffer1/10 total volume2μL
    Diluted BSA (10x)1/10 total volume2μL
    DNAcalculate for target ng3.7μL
    ddH2Ovolume up to (total volume - 1μL)11.3μL
    Enzymegenerally 1μL1μL
    Total Volume20μL
  4. Incubate 60min 37°C.
  5. Heat inactive 70°C for 15 min.
  6. Store in -20°C or use immediately.


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TdT Heat Inactivation



TdT Nucleotide Addition


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


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Yeast Gene Knockout



Yeast Mating



Yeast Sporulation/Zymolyase Treatment



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