"
Team:KIT-Kyoto/Notebook/Protocol
From 2013.igem.org
Miniprep
Solution Ⅰ (50 mM Tris-HCl and 10 mM EDTA, and 50 µg/mL RNase A, pH 8.0 (25˚C))
Solution Ⅱ (0.2 M NaOH and 1 % SDS)
Solution Ⅲ (4 M guanidine hydrochloride and 0.5 M potassium acetate, pH 4.2)
Solution Ⅳ (5 M guanidine hydrochloride, 20 mM Tris-HCl, and 38% ethanol, pH 6.6 (25˚C))
Solution Ⅴ (2 mM Tris-HCl and 80% ethanol, pH 7.5 (25˚C))
カラムなどのメーカーを記載
Pick up a single colony from plate and cultivate it overnight in 3ml LB medium containing appropriate antibiotic at 37˚C˚.
↓
Transfer the culture into a 1.5ml tube, centrifuge at 13,000 rpm for 1 minute, and discard the supernatant (2 times).
↓
Add 250 µL of SolutionⅠ to the pellet and mix well with vortex.
↓
Add 250 µL of SolutionⅡ and mix well by turning the tube upside down several times.
↓
Add 350 µL of SolutionⅢ and by turning the tube upside down several times.
↓
Centrifuge at 13,000 rpm for 5 minutes.
↓
Transfer the supernatant (approx. 850µL) to a mini prep column.
↓
Centrifuge at 13,000 rpm for 1 minute and discard the flow-through.
↓
Add 500 µL of SolutionⅣ.
↓
Centrifuge at 13,000 rpm for 1 minute and discard the flow-through.
↓
Add 700 µL of SolutionⅤ.
↓
Centrifuge at 13,000 rpm for 1 minute and discard the flow-through (2 times).
↓
Set the column on a new 1.5ml tube and add 100 µL of nuclease-free water.
↓
Centrifuge at 13,000 rpm for 1 minute and collect plasmid DNA.
Rapid check of the insert by colony cracking
Add 45 µL of cracking solution into each tube. Cracking solの組成は？？？？
↓
Suspend a small quantity of the colony in a tube using a sterilized toothpick.
↓
Incubate the tube at 65℃ for 10 minutes.
↓
Add a drop of 10x Loading Buffer.
↓
Add equal volume of phenol/chloroform.
↓
Mix with vortex.
↓
Centrifuge at 13,000rpm for 3 minutes.
↓
Apply the supernatant to 1% agarose gel electrophoresis.
↓
Stain the gel in ethidium bromide solution for 10 minutes.
↓
Plasmid DNA can be detected by UV-illuminator as a band between genomic DNA band and low molecular size RNAs.
濃縮精製 DNA purification and precipitation
Mix 350 µL of sterilized water and 50 µL of 3 M sodium acetate with DNA sample.
Mix DNA solution with equal volume of phenol/chloroform by vortex.
Centrifuge at 13,000 rpm for 5 min, then transfer the supernatant into a new tube.
Add equal volume of 2-propanol, and mix by turning the tube upside down.
Centrifuge at 14,500 rpm for 10 min at 4˚C.
Carefully decant the supernatant.
Add adequate volume of 70 % ethanol.
Centrifuge at 14,500 rpm for 5 min at 4˚C.
Carefully decant the supernatant.
Dry in the desiccator under vacuum for 5 min.
You can get dried DNA pellet.
ブルーゲルなどのメーカーを記載
PCR
Adjust the concentration of each primer to 100 pmol/µL with sterilized water.
Mix 10 µL of forward and reverse primer solutions with 80 µL H2O in a new tube (final primer concentration is 10 pmol/µL).
Use 1 µL of primer mix for PCR.
Recipe for PRC is as follows:
|
Buffer |
50 µL |
|
dNTP |
20 µL |
|
Primer mix |
1 µL |
|
DNA sample |
0.5 µL |
|
KOD-FX |
2 µL |
|
H2O |
26.5 µL |
|
total |
100 µL |
KOD-FXなどは(メーカー？？？)
SDS polyacrylamide gel electrophoresis (SDS-PAGE)
12.5% separation gel (recipe for a sheet of gel)
Mili Q water 2.59 ml
acrylamide solution(????? %) 3.33 ml
0.5M Tris (pH8.8) 2 ml
10%SDS 80 µL
10%APS 27 µL
TEMED 4 µL
↓
Apply the acrylamide solution mix to the PAGE glass plate.
↓
Deposit H2O carefully on the top of the acrylamide solution mix.
↓
Wait for 10 min for polymerization.
Stacking gel
Mili Q water 2.89 ml
Acrylamide 0.79 ml
0.5M Tris (pH 6.8) 1.25 ml
10% SDS 50 µL
10%APS 17 µL
TEMED 5 µL
↓
After the polymerization of the separation gel, remove the H2O of the top layer.
↓
Apply stacking gel mix on the running gel and put the comb to make wells.
↓
After the stacking gel has fully polymerized, remove the comb and rinse the top of the gel with H2O and then remove H2O.
Sample preparation
Collect bacterial cells.
↓
Add 450ul of H2O and 50 µL of FastBreak Cell Lysis Reagent (Madison, Wisconsin, USA Promega).
↓
Mix them for 15minutes with shaker.
↓
Add 100ul of 6ｘSDS-Sample buffer and mix with vortex.
↓
Heat samples in a heating block at 99℃ for 5 minutes.
Running samples
Set the gel on the electrophoresis apparatus and apply SDS running buffer (組成は？？？)
↓
Apply the samples and markers.
↓
Operate at constant current (25mA) for 65 minutes per sheet of gel.
Staining
Place the gel in stacking solution( 組成は？？？) and shake gently it for 5 minutes.
↓
Remove the stacking solution and add 100ml of staining solution (組成は？？？).
Warm the gel for 1 minute with a microwave (500W).
↓
Remove the staining solution and rinse the gel with water.
青色ゲル切り出し後の精製
Isolation and purification of DNA bands
Clip DNA band from agarose gel and transfer it into a 1.5 ml tube.
Add H2O. Melt the gel at 65˚C in a heating block.
Add equal volume of phenol. Mix well with vortex.
Centrifuge at 13,000 rpm for 5 min.
Transfer the supernatant into a new tube and mix with equal volume of phenol/chloroform.
Perform the same method of DNA precipitation using 2-propanol.
<ISOLATION OF COLONIES> これは何の実験だ？？？？？
[Procedure]
・Using a toothpick, touch the E. coli growing within the punctured area.
・Streaking for a Single Colony
1) Using the toothpick, gently spread E.coli over a section of the plate, as shown in the diagram, to create streak #1.
2) Using a fresh toothpick, drag through streak #1 and spread E.coli over a second section of the plate, to create streak #2.
3) Using a third toothpick, drag through streak #2 and spread E.coli over the last section of the plate, to create streak #3.
<Ligation>
・Dissolve the purified and dried insert DNA in 5 µL of H2O.
・Dissolve the purified and dried vector DNA in 5μL of H2O.
・Mix the two solutions.
・Add 10μL of Ligation Mix (メーカー？？？) to it.
・Incubate for 15 minutes at room temperature.
<Transformation>
・Add the ligation solution to competent cells (in a clean bench).
・Place tubes on ice for 20 minutes.
・Heat shock treatment at 42℃ for 35 seconds.
・Place tubes on ice for 2 minutes.
・Add 1mL of LB medium into the tube (in a clean bench).
・Incubate the samples for 20 minutes at 37℃.
・Centrifuge at 7,000rpm for 3 minutes.
・Discard the most of supernatant, and spread the remaining cells and LD medium in the tube on the plates (in a clean bench).
・Incubate plates overnight at 37℃.
