Team:KIT-Kyoto/Notebook/Protocol

SDS polyacrylamide gel electrophoresis (SDS-PAGE)

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

・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

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

・Mix them for 15minutes with shaker.

・Add 100µL 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 in 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

1xTAE	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.

Retrieved from "http://2013.igem.org/Team:KIT-Kyoto/Notebook/Protocol"

• Recent changes
• What links here
• Related changes
• Special pages
• My preferences

• Printable version
• Permanent link
• Privacy policy
• Disclaimers