Team:KIT-Kyoto/nf0812

Protocol

• 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))

Pick up a single colony from plate and cultivate it overnight in 3ml LB medium containing appropriate antibiotic at 37˚C˚.

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Transfer the culture into a 1.5ml tube, centrifuge at 13,000 rpm for 1 minute, and discard the supernatant (2 times).

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Add 250 µL of SolutionⅠ to the pellet and mix well with vortex.

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Add 250 µL of SolutionⅡ and mix well by turning the tube upside down several times.

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Add 350 µL of SolutionⅢ and by turning the tube upside down several times.

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Centrifuge at 13,000 rpm for 5 minutes.

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Transfer the supernatant (approx. 850µL) to a mini prep column.

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Centrifuge at 13,000 rpm for 1 minute and discard the flow-through.

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Add 500 µL of SolutionⅣ.

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Centrifuge at 13,000 rpm for 1 minute and discard the flow-through.

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Add 700 µL of SolutionⅤ.

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Centrifuge at 13,000 rpm for 1 minute and discard the flow-through (2 times).

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Set the column on a new 1.5ml tube and add 100 µL of nuclease-free water.

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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(3% w/v SDS ,50mM Tris -base ,pH12.6) into each tube.

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Suspend a small quantity of the colony in a tube using a sterilized toothpick.

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Incubate the tube at 65°C for 10 minutes.

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Add a drop of 10x Loading Buffer.

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Add equal volume of phenol/chloroform.

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Mix with vortex.

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Centrifuge at 13,000rpm for 3 minutes.

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Apply the supernatant to 1% agarose gel electrophoresis.

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Stain the gel in ethidium bromide solution for 10 minutes.

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Plasmid DNA can be detected by UV-illuminator as a band between genomic DNA band and low molecular size RNAs.

• DNA purification and precipitation

1. Mix 350 µL of sterilized water and 50 µL of 3 M sodium acetate with DNA sample.

2. Mix DNA solution with equal volume of phenol/chloroform by vortex.

3. Centrifuge at 13,000 rpm for 5 min, then transfer the supernatant into a new tube.

4. Add equal volume of 2-propanol, and mix by turning the tube upside down.

5. Centrifuge at 14,500 rpm for 10 min at 4˚C.

6. Carefully decant the supernatant.

7. Add adequate volume of 70 % ethanol.

8. Centrifuge at 14,500 rpm for 5 min at 4˚C.

9. Carefully decant the supernatant.

10. Dry in the desiccator under vacuum for 5 min.

11. You can get dried DNA pellet.

• PCR

1. Adjust the concentration of each primer to 100 pmol/µL with sterilized water.

2. Mix 10µL of forward and reverse primer solutions with 80 µL H₂O in a new tube (final primer concentration is 10 pmol/µL).

3. 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

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(Toyobo)

• SDS polyacrylamide gel electrophoresis (SDS-PAGE)

12.5% separation gel (recipe for a sheet of gel)

Mili Q water 2.59 ml

acrylamide solution(30 %) 3.33 ml

0.5M Tris (pH8.8) 2 ml

10%SDS 80 µL

10%APS 27 µL

TEMED 4 µL

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Apply the acrylamide solution mix to the PAGE glass plate.

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Deposit H₂O carefully on the top of the acrylamide solution mix.

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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

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After the polymerization of the separation gel, remove the H₂O of the top layer.

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Apply stacking gel mix on the running gel and put the comb to make wells.

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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.

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Add 450ul of H₂O and 50 µL of FastBreak Cell Lysis Reagent (Madison, Wisconsin, USA Promega).

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Mix them for 15minutes with shaker.

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Add 100ul of 6ｘSDS-Sample buffer and mix with vortex.

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Heat samples in a heating block at 99°C for 5 minutes.

Running samples

Set the gel on the electrophoresis apparatus and apply SDS running buffer (Tris 25mM,Glysine 191mM,SDS0.1%)

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Apply the samples and markers.

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Operate at constant current (25mA) for 65 minutes per sheet of gel.

Staining

Place the gel in stacking solution( 50％ethanol,10％acetic acid) and shake gently it for 5 minutes.

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Remove the stacking solution and add 100ml of staining solution (0.25% CBB R250、5%methanol、7.5%acetic acid).

Warm the gel for 1 minute with a microwave (500W).

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Remove the staining solution and rinse the gel with water.

• Isolation and purification of DNA bands

1. Clip DNA band from agarose gel and transfer it into a 1.5 ml tube.

2. Add H₂O. Melt the gel at 65˚C in a heating block.

3. Add equal volume of phenol. Mix well with vortex.

4. Centrifuge at 13,000 rpm for 5 min.

5. Transfer the supernatant into a new tube and mix with equal volume of phenol/chloroform.

6. Perform the same method of DNA precipitation using 2-propanol.

   
   Blue gel
   ・50×TAE 100ml
   ・Agarose(nacalai tesque) 1.0g
   ・Gel indicator(Biodynamics laboratory) 200ul
   

• Ligation

・Dissolve the purified and dried insert DNA in 5µL of H₂O.

・Dissolve the purified and dried vector DNA in 5µL of H2O.

・Mix the two solutions.

・Add 10µL of Ligation Mix (Wako) 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°C 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°C.

・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°C.