Team:KIT-Kyoto/Notebook/Protocol

This page lists all the protocols used in our project.

LB medium

bacto tryptone	10 g
bacto yeast evtract	5 g
NaCl	10 g

LB plate

LB medium
bacto-agar	15 g/l

↓Add 15g of bacto-agar to 1L of LB medium then sterilize it by autoclave.

↓If necessary, the antibiotics can be added after the temperature of LB-agar become below 65°C.

↓Twenty to twenty-five ml of LB-agar should be poured to the disposable sterilized dishes.

Transformation

↓Dissolve the competent cells on ice.

↓Prepare some iced 1.5ml tubes and pour competent cells for 100µl into the tubes.

↓Add 1~5µl of DNA solution to the 100µl of competent cells, then incubate for 30 minutes on ice.

↓Heat shock for 45 seconds at 42°C.

↓Add 900 µl of SOC medium into each tube.

↓Incubate with shaking at 37°C for 1 hour.

↓Then spread on the LB plate containing an appropriate antibiotic.

↓Incubate at 37°C for 16 hours.

Alkali SDS

↓Spread E.coli onto the LB plate (+amp),and incubate it at 37°C(API2-MALT1 for 30°C) for 16 hours.

↓Pick up the single colony from the plate.

↓Incubate it into the 2ml LB medium, and shake the tubes at 37°C(API2-MALT1 for 30°C) for hours.

↓Harvest the bacterial cells by centrifugation at 4°C for 5 minutes (5,000 x g).Discard the supernatant.

↓Add 100µl of iced SolutionⅠ to the tubes,and mix them using vortex.

↓Add 200µl of iced SolutionⅡ to the tubes,and mix them sharply not using vortex.

↓Let them stand on ice for 5 minutes.

↓Add 150µl of iced Solution Ⅲ to the tubes,and mix them sharply but not using vortex.

↓Let them stand on ice for 5 minutes.

↓Centrifuge them at 4°C for 10 minutes (12,000 x g).

↓Transfer supernatant into the tubes.

↓Add 450µl of Isopropanol and mix them well.Let them for 15 minutes.Then recover the DNA by centrifugation at 12,000 x g for 30 minutes at 25°C.

Ligation

Refer to following table, prepare a reaction solution.

insert	0.5 µl
vector	0.5 µl
2 x Buffer	2.5 µl
T4 ligase	0.5 µl
H2O	1.0 µl
	total 5 µl

Incubate it for 30min at 16 ℃.

PCR

Add all reagents in a PCR tube.

Based on primers, set an appropriate annealing temperature.

agarose gel electrophoresis

↓Prepare a 1% (w/v) agarose gel.

↓Add 10 µl of 6x loading dye to each 50 µl of digested solutions.

↓Load each of 60 µL samples and DNA maker in wells.

↓Run at 50 V and 60min.

↓Stain in EtBr solution for 10min.

Making a coptent cells

↓Streak E.coli on LB plate and incubate for 16h.

↓Pick a single colony up, transfer it into 250 ml of SOB medium in a flask. Incubate it at 18°C with shaking.

↓When it reaches an OD600 of 0.4 to 0.8, transfer it on an ice for 10 min.

↓Harvest cells by centrifugation(4.5 x 10³ g) for 10min at 4°C.

↓Remove the supernatant and resuspend the cells in 84 ml of ice-cold TB.

↓Keep it on ice for 10min.

↓Harvest cells by centrifugation(5 x 10³ g) for 10min at 4°C.

↓Remove the supernatant and resuspend the cells in 40 ml of ice-cold TB.

↓Add 1.5 ml of DMSO(7%) and keep it on ice for 10min.

↓Pour each of 220 µl into chilled eppendorf tubes. Make sure to close tightly and then flash freeze in liquid nitrogen and store at -80°C.

Transformation Buffer(TB)

↓1.5 g of 1M PIPES Buffer

↓1.1 g of CaCl₂H₂O

↓9.3 g KCl

↓Bring to volume 400 mL; pH to 6.7 with 5M KOH

↓Add 5.45 g MnCl₂4H₂O

↓Bring volume to 500mL and sterile filter into sterile bottle.