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− | <div class="column full_size">
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− | <p> Document the dates you worked on your project.</p>
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− | <h5>What should this page have?</h5>
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− | <li>Chronological notes of what your team is doing.</li>
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− | <li> Brief descriptions of daily important events.</li>
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− | <li>Pictures of your progress. </li>
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− | <li>Mention who participated in what task.</li>
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− | <h5>Inspiration</h5>
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− | <p>You can see what others teams have done to organize their notes:</p>
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− | <li><a href="https://2014.igem.org/Team:ATOMS-Turkiye/Notebook">2014 ATOMS-Turkiye</a></li>
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− | <li><a href="https://2014.igem.org/Team:Tec-Monterrey/ITESM14_project.html#tab_notebook">2014 Tec Monterrey</a></li>
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− | <li><a href="https://2014.igem.org/Team:Kyoto/Notebook/Magnetosome_Formation#title">2014 Kyoto</a></li>
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− | <li><a href="https://2014.igem.org/Team:Cornell/notebook">2014 Cornell</a></li>
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− | </ul>
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| <h1>Protocols</h1> | | <h1>Protocols</h1> |
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Revision as of 03:08, 17 August 2016
Protocols
PCR
Use KOD -Plus- Neo (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 10µM |
Primer-R 10µM |
MgSO4 |
dNTPs |
10x Buffer |
KOD Plus Neo |
DW |
Total |
Volume (µL) |
1 |
1 |
1 |
3 |
5 |
5 |
1 |
33 |
50 |
Thermal protocol is following
2STEP Cycle (Tm value > 63°C)
Sequence | Temp. (°C) | Time (sec) |
1 | 94 | 120 |
2 | 98 | 10 |
3 | 68 | 30sec / 1kbp |
4 | 4 | Hold |
Cycle: sequence2~3 × (25~45)
3STEP Cycle (Tm value < 63°C)
Sequence | Temp. (°C) | Time (sec) |
1 | 94 | 120 |
2 | 98 | 10 |
3 | Tm | 30 |
4 | 68 | 30sec / 1kbp |
5 | 4 | Hold |
Cycle: sequence2~4 × (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 10U/µL |
RE2 10U/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&rt; (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.
- Pore 2x TBE buffer into the tank to soak gel.
- Add 5 µL of EtBr into cathod.
- Pre-migration for 30 min at 100 V.
- Apply DNA solution with 6x loading dye and ladder.
- Start electrophoresis at 100 V.
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.
- Leave it at room temperature for 5 min.
- Centrifuge at 15,000 rpm for 15 min at 25°C.
- Remove supernatant and add 600 µL of 70% ethanol.
- Centrifuge at 15,000 rpm for 5 min at 25°C.
- Remove supernatant and air-dry at room temperature with light sheilding.
- Suspend with 10 µL of TE.
Colony PCR
Solution |
DNA |
Kapa-Taq (Taq polymerase) |
EX-F primer 10µM |
PS-R primer 10µM |
Total |
Volume (µL) |
4.2 |
5 |
0.4 |
0.4 |
10 |
Sequence | Temp. (°C) | Time (sec) |
1 | 94 | 120 |
2 | 94 | 30 |
3 | 68 | 60 sec / 1kbp |
4 | 4 | Hold |
cycles: sequence2~3 × 25~45
Sequencing
Solution |
5 x Sequencing Buffer |
primer 1µM |
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: sequence2~4 × 25
Ethanol precipitation
Solution |
PCR product |
DW |
3M NaOAc |
Glycogen |
100% EtOH |
Volume (µL) |
10 |
10 |
2 |
1 |
50 |
- Centrifuge at 15,000 rpm for 15 min at room temprature
- Remove supernatant ,add 100 µL of 70% EtOH and tap tubes by finger.
- Centrifuge at 15,000 rpm for 10 min at room temprature
- Remove supernatant and air dry at room temperature.
- Resuspend the pellet to HiDi formamide and remove to 96-well plate.
- Set the plate and start electrophoresis.
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 differnt 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
Streaking (Single colony isolation)
- Pick the colony with an inoculating loop from the agar plate.
- Drag the loop across on a new agar plate.
- Re-sterilise the loop and drag it across again.
- Repeat method 3.
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 air-dry at room temperature with light sheilding.
- Suspend with 10 µL of TE.
Gel Free System
Preparation of biotinylated DNA fragments
Use KOD -Plus- Neo (TOYOBO CO.,LTD) as polymerase and 5'-biotinylated primers. Mix PCR solutions and run the PCR machine in a program which is detailed below.
Solution |
template DNA |
5'-biotinylated 100-UP primer 10µM |
5'-biotinylated 200-DN primer 10µM |
MgSO4 |
dNTPs |
10x Buffer |
KOD Plus Neo |
DW |
Total |
Volume (µL) |
1 |
1.5 |
1.5 |
3 |
5 |
5 |
1 |
32 |
50 |
Thermal protocol is following
2STEP Cycle (Tm value > 63°C)
Sequence | Temp. (°C) | Time (sec) |
1 | 94 | 120 |
2 | 98 | 10 |
3 | 68 | 30sec / 1kbp |
4 | 4 | Hold |
Cycle: sequence2~3 × (25~45)
Preparation of magnetic beads
- Mix 2 µL of magnetic beads (SiMAG-Streptavidin) and 48 µL of TE by vibration using sonic-toothbrush.
- Collect the beads by attracting them to one side in 0.2 mL polypropylene tube using neodymium magnet.
- Remove supernatant.
-
Fixation to magnetic beads
- Add 3 µL of PCR product (0.48 pmol) and 7 µL of TE to beads.
- Mix by vibration using sonic-toothbrush.
- Collect the beads using magnet.
- Remove supernatant containing excess amount of free DNA fragment.
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.