Team:KIT-Kyoto/Notebook/Protocol

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(Difference between revisions)
Line 45: Line 45:
:↓Add 1~5µl of DNA solution to the 100µl of competent cells, then incubate for 30 minutes on ice.
:↓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.
:↓Heat shock for 45 seconds at 42°C.
-
:↓Add 900 µl of soc medium into each tube.
+
:↓Add 900 µl of SOC medium into each tube.
:↓Incubate with shaking at 37°C for 1 hour.
:↓Incubate with shaking at 37°C for 1 hour.
:↓Then spread on the LB plate containing an appropriate antibiotic.
:↓Then spread on the LB plate containing an appropriate antibiotic.

Revision as of 03:01, 6 October 2011



Home Team Project Parts Notebook Safety Human Practice Attributions


Home > Notebook > Protocol

This page lists all the protocols used in our project.

LB medium

bacto tryptone10 g
bacto yeast evtract5 g
NaCl10 g



LB plate

LB medium 
bacto-agar15 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.
insert0.5 µl
vector0.5 µl
2 x Buffer2.5 µl
T4 ligase0.5 µl
H2O1.0 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)

PIPES1.5 g
CaCl2H2O1.1 g
KCl9.3 g
↓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.