Experiments
In addition to standard experiments as protocols below show, we conducted SDS-PAGE to find that we could make correct proteins about BBa_K2414002 and BBa_K2414003, and analyzed activity of Phytase ,which are existing parts BBa_K912000, BBa_K912001, and new parts BBa_K2414002, and BBa_K2414003. See Results.
We analyzed all the Phytases with this protocol below:
Protocol: 50μl 7.5 mM Phytic acid 190μl CH3COOH Buffer
Digest phytic acid in above situation with sample phytase solution 10μl, 37degrees celcius, 10 min.
After digestion, using AMM solution (2ml of 10mM NH4Mo7O2.4H2O: 5N H2SO4: Acetone= 1:1:2) analyze Optical Absorbance. The more Pi are there, the more yellow AMM becomes.
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)
| Sequence | Temp. (°C) | Time (sec) |
| 1 | 94 | 120 |
| 2 | 98 | 10 |
| 3 | 68 | 30 / 1kbp |
| 4 | 4 | Hold |
Cycle: sequence 2~3 × (25~45)
3STEP Cycle (Tm value < 68°C)
| Sequence | Temp. (°C) | Time (sec) |
| 1 | 94 | 120 |
| 2 | 98 | 10 |
| 3 | Tm | 30 |
| 4 | 68 | 30 / 1kbp |
| 5 | 4 | Hold |
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 |
| Sequence | Temp. (°C) | Time (min) |
| 1 | 37 | 120 |
| 2 | 65 | 15 |
| 3 | 4 | Hold |
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.
| Solution | Vector DNA | Insert DNA | DW | Mighty Mix | Total |
| Volume (µL) | 1 | 2 | 2 | 5 | 10 |
Thermal protocol is following
| Sequence | Temp. (°C) | Time (min) |
| 1 | 16 | 30 |
| 2 | 65 | 10 |
| 3 | 4 | Hold |
Electrophoresis
- Put gel into electrophoresis tank.
- Pour 1x TAE buffer into the tank to soak gel.
- Pre-migration for 30 min at 100 V.
- Apply DNA solutions with 6x loading dye and ladder.
- Start electrophoresis at 100 V.
- Stop at appropriate time.
- 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
- Add 1/10 volume of NaOAc, and 5/2 of 100% ethanol.
- Store at -20°C for 10 min.
- Centrifuge at 15,000 rpm for 15 min at 4°C.
- Remove supernatant and add 500 µL of 70% ethanol.
- Centrifuge at 15,000 rpm for 5 min at 4°C.
- Remove supernatant and dry up at room temperature using vacuum drier.
- 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 |
| Sequence | Temp. (°C) | Time (sec) |
| 1 | 94 | 300 |
| 2 | 98 | 10 |
| 3 | 68 | 60 / 1kbp |
| 4 | 4 | Hold |
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 |
| Sequence | Temp. (°C) | Time (sec) |
| 1 | 96 | 10 |
| 2 | 50 | 5 |
| 3 | 60 | 240 |
| 4 | 4 | Hold |
Cycle: sequence 2~4 × 25
Competent Cells
- Thaw original competent cells on ice.
- Add 5 µL of original competent cells to 2 mL of LB.
- Incubate the cells for 16 hrs at 37°C.
- Add 5 µL, 50 µL, and 500 µL of original cells to 100 mL of LB.
- Incubate the cells at 130 rpm at 20°C, until OD600 reach 0.5.
- Take 50 mL of incubated cells to two different culture tubes and centrifuge them at 3,000 rpm for 20 min at 4°C.
- Remove supernatant and add 75 mL of TB to each tube.
- Bring them to a one tube and centrifuge at 3,000 rpm for 20 min at 4°C.
- Remove supernatant and add 32 mL of TB.
- Add 32 µL of DMSO 10 times.
- Take 50 µL and freeze with liquid nitrogen.
Transformation
- Add plasmid to thawed competent cells on ice.
- Incubate on ice for 30 min.
- Add to LB.
- Incubate the cells for 2 hrs at 37°C.
- Spread 300 µL of the culture onto plate with LB and appropriate antibiotics.
- 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
- Add 13 µL of PEG to 20 µL of product(s).
- Leave it at room temperature for 1 hr.
- Centrifuge at 15,000 rpm for 20 min at 4°C.
- Remove supernatant and add 100 µL of 70% ethanol.
- Centrifuge at 15,000 rpm for 2 min at 4°C.
- Remove supernatant and dry up at room temperature with light sheilding.
- Suspend with 10 µL of TE.
Double restriction digestion
- 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.
- Mix by pumping using pipette.
- Incubate at 37 °C for 30 min.
- Collect the beads using magnet.
- Obtain supernatant containing digested DNA fragment.
- Purify the supernatant by ethanol precipitation.
