Line 80: | Line 80: | ||
Cycle: sequence2~3 × (25~45) | Cycle: sequence2~3 × (25~45) | ||
− | < | + | <h2>3STEP Cycle (Tm value < 63°C)</h2> |
<table> | <table> | ||
<tr> | <tr> | ||
Line 103: | Line 103: | ||
Cycle: sequence2~4 × (25~45) | Cycle: sequence2~4 × (25~45) | ||
− | < | + | <h2>STEP DOWN</h2> |
<table> | <table> | ||
<tr> | <tr> | ||
Line 172: | Line 172: | ||
− | < | + | <h1 onClick="hyoji2()"><span class="ka-soru">PCR Purification</span></h1> |
<div id="disp2"> | <div id="disp2"> | ||
Line 199: | Line 199: | ||
</script> | </script> | ||
− | < | + | <h1 onClick="hyoji3()"><span class="ka-soru">Digestion</span></h1> |
<div id="disp3"> | <div id="disp3"> | ||
<span class="nomal2"> | <span class="nomal2"> | ||
Line 257: | Line 257: | ||
− | < | + | <h1 onClick="hyoji4()"><span class="ka-soru">Ligation</span></h1> |
<div id="disp4"> | <div id="disp4"> | ||
<span class="nomal2"> | <span class="nomal2"> | ||
Line 305: | Line 305: | ||
</script> | </script> | ||
− | < | + | <h1 onClick="hyoji5()"><span class="ka-soru">Electrophoresis</span></h1> |
<div id="disp5"> | <div id="disp5"> | ||
<span class="nomal2"> | <span class="nomal2"> | ||
Line 338: | Line 338: | ||
− | < | + | <h1 onClick="hyoji6()"><span class="ka-soru">Gel Extraction</span></h1> |
<div id="disp6"> | <div id="disp6"> | ||
<span class="nomal2"> | <span class="nomal2"> | ||
Line 365: | Line 365: | ||
− | < | + | <h1 onClick="hyoji7()"><span class="ka-soru">Ethanol precipitation</span></h1> |
<div id="disp7"> | <div id="disp7"> | ||
<span class="nomal2"> | <span class="nomal2"> | ||
Line 398: | Line 398: | ||
</script> | </script> | ||
− | < | + | <h1 onClick="hyoji8()"><span class="ka-soru">Colony PCR</span></h1> |
<div id="disp8"> | <div id="disp8"> | ||
<span class="nomal2"> | <span class="nomal2"> | ||
Line 458: | Line 458: | ||
</script> | </script> | ||
− | < | + | <h1 onClick="hyoji9()"><span class="ka-soru">Sequencing</span></h1> |
<div id="disp9"> | <div id="disp9"> | ||
<span class="nomal2"> | <span class="nomal2"> | ||
Line 502: | Line 502: | ||
− | < | + | <h2>Ethanol precipitation</h2> |
<table> | <table> | ||
<tr> | <tr> | ||
Line 550: | Line 550: | ||
− | < | + | <h1 onClick="hyoji10()"><span class="ka-soru">Competent Cells</span></h1> |
<div id="disp10"> | <div id="disp10"> | ||
<span class="nomal2"> | <span class="nomal2"> | ||
Line 586: | Line 586: | ||
</script> | </script> | ||
− | < | + | <h1 onClick="hyoji11()"><span class="ka-soru">Transformation</span></h1> |
<div id="disp11"> | <div id="disp11"> | ||
<span class="nomal2"> | <span class="nomal2"> | ||
Line 617: | Line 617: | ||
</script> | </script> | ||
− | < | + | <h1 onClick="hyoji12()"><span class="ka-soru">Mini-prep</span></h1> |
<div id="disp12"> | <div id="disp12"> | ||
<span class="nomal2"> | <span class="nomal2"> | ||
Line 642: | Line 642: | ||
</script> | </script> | ||
− | < | + | <h1 onClick="hyoji13()"><span class="ka-soru">Streaking (Single colony isolation)</span></h1> |
<div id="disp13"> | <div id="disp13"> | ||
<span class="nomal2"> | <span class="nomal2"> | ||
Line 671: | Line 671: | ||
</script> | </script> | ||
− | < | + | <h1 onClick="hyoji14()"><span class="ka-soru">PEG precipitation</span></h1> |
<div id="disp14"> | <div id="disp14"> | ||
<span class="nomal2"> | <span class="nomal2"> | ||
Line 703: | Line 703: | ||
</script> | </script> | ||
− | < | + | <h1 onClick="hyoji15()"><span class="ka-soru">Gel Free System</span></h1> |
<div id="disp15"> | <div id="disp15"> | ||
<span class="nomal2"> | <span class="nomal2"> | ||
− | < | + | <h2>Preparation of biotinylated DNA fragments</h2> |
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. | 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. | ||
Line 736: | Line 736: | ||
</table> | </table> | ||
Thermal protocol is following | Thermal protocol is following | ||
− | < | + | <h2>2STEP Cycle (Tm value > 63°C)</h2> |
<table> | <table> | ||
<tr> | <tr> | ||
Line 756: | Line 756: | ||
Cycle: sequence2~3 × (25~45) | Cycle: sequence2~3 × (25~45) | ||
− | < | + | <h2>Preparation of magnetic beads</h2> |
<ol> | <ol> | ||
<li>Mix 2 µL of magnetic beads (SiMAG-Streptavidin) and 48 µL of TE by vibration using sonic-toothbrush.</li> | <li>Mix 2 µL of magnetic beads (SiMAG-Streptavidin) and 48 µL of TE by vibration using sonic-toothbrush.</li> | ||
Line 763: | Line 763: | ||
</ol> | </ol> | ||
− | < | + | <h2>Fixation to magnetic beads</h2> |
<ol> | <ol> | ||
<li>Add 3 µL of PCR product (0.48 pmol) and 7 µL of TE to beads.</li> | <li>Add 3 µL of PCR product (0.48 pmol) and 7 µL of TE to beads.</li> | ||
Line 771: | Line 771: | ||
</ol> | </ol> | ||
− | < | + | <h2>Double restriction digestion</h2> |
<ol> | <ol> | ||
<li>Add Digestion Premix containing 1 µL of 10x RE solution, 8 µL of DW and each 0.5 µL of restriction endonuclease, <I>Xba</I>I and <I>Spe</I>I, to the beads.</li> | <li>Add Digestion Premix containing 1 µL of 10x RE solution, 8 µL of DW and each 0.5 µL of restriction endonuclease, <I>Xba</I>I and <I>Spe</I>I, to the beads.</li> |
Revision as of 11:33, 17 October 2016
Team:HokkaidoU Japan
\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
Cycles: sequence2~3 × 25~45
Cycle: sequence2~4 × 25
Thermal protocol is following
Cycle: sequence2~3 × (25~45)
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.
Thermal protocol is following
Cycle: sequence2~3 × (25~45)
Cycle: sequence2~4 × (25~45)
Cycle:
sequence2~3 × 5
sequence4~5 × 5
sequence6~7 × 5
sequence8~9 × 15
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 |
2STEP Cycle (Tm value > 63°C)
Sequence | Temp. (°C) | Time (sec) |
---|---|---|
1 | 94 | 120 |
2 | 98 | 10 |
3 | 68 | 30sec / 1kbp |
4 | 4 | Hold |
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 |
STEP DOWN
Sequence | Temp. (°C) | Time (sec) |
---|---|---|
1 | 94 | 120 |
2 | 98 | 10 |
3 | 74 | 30sec / 1kbp |
4 | 98 | 10 |
5 | 72 | 30 |
6 | 98 | 10 |
7 | 70 | 30 |
8 | 98 | 10 |
9 | 68 | 30 |
10 | 68 | 420 |
11 | 4 | Hold |
sequence2~3 × 5
sequence4~5 × 5
sequence6~7 × 5
sequence8~9 × 15
PCR Purification
FastGeneTM Gel/PCR Extraction kit (Nippon Genetics Co., Ltd)
Purification of PCR products
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.
Thermal protocol is following
Solution | Vector DNA | Insert DNA | DW | Mighty Mix | Total |
---|---|---|---|---|---|
Volume (µL) | 1 | 2 | 2 | 5 | 10 |
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
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 |
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 |
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
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 |
2STEP Cycle (Tm value > 63°C)
Sequence | Temp. (°C) | Time (sec) |
---|---|---|
1 | 94 | 120 |
2 | 98 | 10 |
3 | 68 | 30sec / 1kbp |
4 | 4 | Hold |
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.