Team:KIT-Kyoto/Notebook/Protocol

Miniprep

Solution Ⅰ (50 mM Tris-HCl and 10 mM ED TA, 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˚.

・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(3% w/v SDS ,50mM Tris -base ,pH12.6) into each tube. ・Suspend a small quantity of the colony in a tube using a sterilized toothpick. ・Incubate the tube at 65°C 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.

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.

PCR

・Adjust the concentration of each primer to 100 pmol/µL with sterilized water. ・Mix 10µL 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

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

・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(pH6.8)	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°C for 5 minutes.

Running samples

・Set the gel on the electrophoresis apparatus and apply SDS running buffer (Tris 25mM,Glysine 191mM,SDS0.1%) ・Apply the samples and markers. ・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. ・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). ・Remove the staining solution and rinse the gel with water.

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.

Blue gel

50×TAE	100mL
Agarose(Nacalai tesque)	1.0g
Gel indicator(Biodynamics laboratory)	200 µL

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.

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