Team:BostonU/Protocols

OUR TEAM

Below is an interactive list of the laboratory techniques employed by the team. Click to expand for details.

Colony PCR

Materials

  • 5% DMSO
  • 2x KOD Hot Start Master Mix
  • PCR Tubes
  • Forward Primers(s) [10 uM]
  • Reverse Primer(s) [10 uM]
  • DNA Template
  • Deionized water

Procedure

  1. Add 18.5 uL of deionized water to PCR tube
  2. Add 1.5 µl of both the requisite Forward and Reverse Primers to PCR tube
  3. Add 1 ul of DNA template
  4. Add 27.5 µl of KOD Hot start Master Mix to PCR tube
  5. Centrifuge for 10 seconds to remove air bubbles
  6. Place in Thermocycler

Thermocycler Conditions

Number of Cycles From 20 - 40
Annealing Temperature Set temperature to the lowest primer melt temperature
Extension Time If target size is:
< 500 bp run 10 sec/kb
500-1000 bp run 15 sec/kb
1000-3000 bp run 20 sec/kb
< 3000 bp run 20 sec/kb

Colony PCR

Materials

  • 5% DMSO
  • 2x KOD Hot Start Master Mix
  • PCR Tubes
  • Forward Primers(s) [10 uM]
  • Reverse Primer(s) [10 uM]
  • DNA Template
  • Deionized water

Procedure

  1. Prepare the DNA template by picking a colony from a plate and mixing it into 10 uL of deionized water in a PCR tube
  2. Add 17.5 µl of deionized water to another PCR tube
  3. Add 1.5 µl of both the requisite Forward and Reverse Primers to the second PCR tube
  4. Add 2 ul of water containing cells with DNA template sample
  5. Add 27.5 µl of KOD Hot Start Master Mix
  6. Centrifuge for 10 seconds to remove air bubbles
  7. Place in Thermocycler

Thermocycler Conditions

Number of Cycles From 20 - 40
Annealing Temperature Set temperature to the lowest primer melt temperature
Extension Time If target size is:
< 500 bp run 10 sec/kb
500-1000 bp run 15 sec/kb
1000-3000 bp run 20 sec/kb
< 3000 bp run 20 sec/kb

Overlap Extension PCR

Materials

  • 5% DMSO
  • 2x KOD Hot Start Master Mix
  • PCR Tubes
  • Forward Primers(s) [10 uM]
  • Reverse Primer(s) [10 uM]
  • DNA Template
  • Deionized water

Procedure

  1. Add equimolar concentrations of each DNA sample to a PCR tube
  2. Add 1.5 uL of each Forward and Revers Primer
  3. Add 27.5 uL of KOD Hot Start Master Mix
  4. Add deionized water until the total volume is 50 uL
  5. Place in Themocycler

Thermocycler Conditions

Number of Cycles From 20 - 40
Annealing Temperature Set temperature to the lowest primer melt temperature
Extension Time If target size is:
< 500 bp run 10 sec/kb
500-1000 bp run 15 sec/kb
1000-3000 bp run 20 sec/kb
< 3000 bp run 20 sec/kb