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 is BBa_K2414000, BBa_K2414001, BBa_K2414002, and BBa_K2414003. See Results.
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.