Team:HokkaidoU Japan/Experiments

Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

What should this page contain?
  • Protocols
  • Experiments
  • Documentation of the development of your project

PCR

Use KOD -Plus (TOYOBO CO.,LTD) as polymerase. Mix PCR solutions and run the PCR machine in a program which is detailed below.
Solution template DNA Primer-F 10 µM Primer-R 10 µM MgSO4 dNTPs 10x Buffer KOD Plus DW Total
Volume (µL) 1 1.5 1.5 3 5 5 1 32 50
Thermal protocol is following

2STEP Cycle (Tm value > 68°C)

SequenceTemp. (°C)Time (sec)
194120
29810
36830 / 1kbp
44Hold
Cycle: sequence 2~3 × (25~45)

3STEP Cycle (Tm value < 68°C)

SequenceTemp. (°C)Time (sec)
194120
29810
3Tm30
46830 / 1kbp
54Hold
Cycle: sequence 2~4 × (25~45)

PCR Purification

FastGeneTM Gel/PCR Extraction kit (Nippon Genetics Co., Ltd)
Purification of PCR products

Digestion

Mix the following reagents in PCR tube.
Solution DNA RE1 10 U/µL RE2 10 U/µL Appropriate buffer Total
Volume (µL) 16 1 1 2 20
SequenceTemp. (°C)Time (min)
137120
26515
34Hold

Ligation

Mix the following reagents in 0.2 mL PCR tube. Use DNA Ligation Kit Mighty Mix (Takara Bio Inc.) which contains ligase and buffer.
SolutionVector DNAInsert DNADWMighty MixTotal
Volume (µL)122510
Thermal protocol is following
SequenceTemp. (°C)Time (min)
11630
26510
34Hold

Electrophoresis

  1. Put gel into electrophoresis tank.
  2. Pour 1x TAE buffer into the tank to soak gel.
  3. Pre-migration for 30 min at 100 V.
  4. Apply DNA solutions with 6x loading dye and ladder.
  5. Start electrophoresis at 100 V.
  6. Stop at appropriate time.
  7. Soak gel in the Ethidium Bromide solution for 30 minute

Gel Extraction

FastGeneTM Gel/PCR Extraction kit (Nippon Genetics Co., Ltd)
DNA extraction from gel

Ethanol precipitation

  1. Add 1/10 volume of NaOAc, and 5/2 of 100% ethanol.
  2. Store at -20°C for 10 min.
  3. Centrifuge at 15,000 rpm for 15 min at 4°C.
  4. Remove supernatant and add 500 µL of 70% ethanol.
  5. Centrifuge at 15,000 rpm for 5 min at 4°C.
  6. Remove supernatant and dry up at room temperature using vacuum drier.
  7. Suspend with 15 µL of TE.

Colony PCR

Solution bacterial suspension Primer-F 10 µM Primer-R 10 µM MgSO4 dNTPs 10x Buffer KOD Plus Total
Volume (µL) 6.6 0.3 0.3 0.6 1 1 0.2 10
Volume (µL) 6.8 5 0.4 0.4 10
SequenceTemp. (°C)Time (sec)
194300
29810
36860 / 1kbp
44Hold
Cycles: sequence 2~3 × 25~45

Sequencing

Solution 5 x Sequencing Buffer primer 1 µM template DNA Ready Reaction Premix DW Total
Volume (µL) 1.5 1.5 1 1 5 10
SequenceTemp. (°C)Time (sec)
19610
2505
360240
44Hold
Cycle: sequence 2~4 × 25

Competent Cells

  1. Thaw original competent cells on ice.
  2. Add 5 µL of original competent cells to 2 mL of LB.
  3. Incubate the cells for 16 hrs at 37°C.
  4. Add 5 µL, 50 µL, and 500 µL of original cells to 100 mL of LB.
  5. Incubate the cells at 130 rpm at 20°C, until OD600 reach 0.5.
  6. Take 50 mL of incubated cells to two different culture tubes and centrifuge them at 3,000 rpm for 20 min at 4°C.
  7. Remove supernatant and add 75 mL of TB to each tube.
  8. Bring them to a one tube and centrifuge at 3,000 rpm for 20 min at 4°C.
  9. Remove supernatant and add 32 mL of TB.
  10. Add 32 µL of DMSO 10 times.
  11. Take 50 µL and freeze with liquid nitrogen.

Transformation

  1. Add plasmid to thawed competent cells on ice.
  2. Incubate on ice for 30 min.
  3. Add to LB.
  4. Incubate the cells for 2 hrs at 37°C.
  5. Spread 300 µL of the culture onto plate with LB and appropriate antibiotics.
  6. Incubate the plate(s) at 37°C for 16~20 hrs.

Mini-prep

FastGeneTM Plasmid mini kit (Nippon Genetics Co., Ltd)
fast / standard / low copy protocol

PEG precipitation

  1. Add 13 µL of PEG to 20 µL of product(s).
  2. Leave it at room temperature for 1 hr.
  3. Centrifuge at 15,000 rpm for 20 min at 4°C.
  4. Remove supernatant and add 100 µL of 70% ethanol.
  5. Centrifuge at 15,000 rpm for 2 min at 4°C.
  6. Remove supernatant and dry up at room temperature with light sheilding.
  7. Suspend with 10 µL of TE.

Double restriction digestion

  1. Add Digestion Premix containing 1 µL of 10x RE solution, 8 µL of DW and each 0.5 µL of restriction endonuclease, XbaI and SpeI, to the beads.
  2. Mix by pumping using pipette.
  3. Incubate at 37 °C for 30 min.
  4. Collect the beads using magnet.
  5. Obtain supernatant containing digested DNA fragment.
  6. Purify the supernatant by ethanol precipitation.