Team:Caltech/Colony PCR

From 2010.igem.org


iGEM 2010



Home

People

Project Details

Protocols

Completed Systems

Notebook

Biosafety

Human Impact

References

Support


Caltech logo watermark.png

Colony PCR

Materials

  • Autoclaved water
  • PCR tubes
  • LB broth & plates
  • Qiagen PCR Core Kit: dNTPs, Taq, Buffers
  • Forward & reverse primers

Procedure

  1. 85.5μL autoclaved water were added to PCR tubes - one for each colony to be amplified.
  2. Inoculate the tube with cells from the desired colony. With the same toothpick, simultaneously inoculate a 5mL LB liquid culture (with antibiotic) and streak an LB-agar plate.
  3. Incubate PCR tubes in a thermal cycler for 10 minutes at 100°C. Remove and cool.
  4. To each tube, add: 10μL 10x PCR buffer, 2μL 10mM dNTP mix, 1μL 10μM forward primer, 1μL 10μM reverse primer, 0.5μL Taq DNA polymerase. Note: it is generally much more efficient to combine all these in a single tube and scale up for multiple reactions.
  5. Replace tubes in thermal cycler and initiate CPCR program (below).
  6. Analyze reactions via 1% agarose gel electrophoresis:
    1. Add 2μL NEB 6x Gel Loading Buffer to 10μL CPCR product.
    2. Load into gel. Run for 30-40 minutes at 90-100V.
    3. Stain/destain in EtBr and image using UV illumination.

Program

  1. Initial denaturation: 94°C, 30s
  2. 30 cycles:
    1. Denaturation: 94°C, 3 min
    2. Annealing: 54°C, 30s
    3. Elongation: 72°C, 2 min
  3. Final extension: 72°C, 2 min
  4. Finish: 5°C hold

Primers

  • Forward: 5'-GAA TTC GCG GCC GCT TCT AGA G-3'
  • Reverse: 5'-CTG CAG CGG CCG CTA CTA GTA-3'
 
Caltech logo.png
Caltech footer.png


Locations of visitors to this page