Making Competent Cells

Step	Method
1	Streak E.coli cells on an LB plate.
2	Allow cells to grow at 37°C overnight.
3	Place one colony in 10 mL LB media(+antibiotic selection if necessary), grow overnight at 37°C.
4	Take 2 ml LB media and save for blank. Transfer 500 µL overnight culture into 50 mL LB media in 250 mL flask.
5	Allow cell to grow at 37°C (200 rpm), until OD600= 0.5 (~2-3 hours).
6	Place cells on ice for 30 minutes.
7	Transfer cells to a centrifuge tube (50 mL), and centrifuge cells at 4°C for 10 minutes at 4,000×g.
8	Pour off media and resuspend cells in 12 mL of cold TB.
9	Centrifuge cells at 4°C for 10 minutes at 4,000×g.
10	Pour supernatant and resuspend cells (by pipetting) in 4 mL of cold TB and 280 µL of DMSO. Transfer 100 µL to 1.5 mL tube.
11	Ependorff tubes placed on ice. Freeze the cells in liquid nitrogen. Cells stored at -80°C can be used for transformation for up to ~6 months.

PCR

1. Mix the following:

Name	Volume
PrimeSTAR® Max DNA Polymerase	10µL
Template DNA	1µL
Primer Forward (10 µM)	1 µL
Primer Reverse (10 µM)	1µL
ddH2O	up to 20µL
Total	20 µL

2. Put samples into Thermal Cyclers and run the following steps: 72 °C

PreDenature	Denature	Anealing	Extension	cycle
98 °C	98 °C	Tm-5 °C	--
2 min	30 sec	30 sec	15 sec /kb	30 cycles

3. Agarose Gel Electrophoresis for confirmation.

Miniprep

Step	Method
1	Pellet 1-4ml of overnight culture by centrifugation at 12,000×g for 1 minute. Discard the supertanant completely.
2	Add 250ul of SolutionⅠto the pellet to resuspend bacteria cells.
3	Add 250ul of SolutionⅡ, mix gently by inverting the tube 7-8 times until the solution becomes clear. The time should be no longer than 5 minutes.
4	Add 350ul of SolutionⅢ, mix gently by inverting the tube 7-8 times.
5	Centrifuge at 12,000rpm for 10 minutes.
6	Place spin column into a 1.5ml collection tube. Transfer supernatant in the step above to the column. Centrifuge at 12,000rpm for 1 minute. Discard the filtrate from the 2ml microfuge tube.
7	Return the column to the 2ml microfuge tube and add 600ul of Buffer PW. Centrifuge at 12,000×g for 1 minute. Discard the filtrate from the 2ml microfuge tube.
8	Return the column to the 2ml microfuge tube and add 600ul of Buffer PW. Centrifuge at 12,000×g for 1 minute. Discard the filtrate from the 2ml microfuge tube.
9	Place the column back into the 2ml microfuge tube. Centrifuge at 12,000×g for 2 minute. Discard the filtrate and the 2ml microfuge tube.
10	Place the spin column onto dry block heater for 8 minute.
11	Transfer the column into a clean 1.5ml microfuge tube .Add 60-80ul of Eluent or deionized water to the center of the membrane to elute the DNA. Let it stand for 1 minute at room temperature. Centrifuge at 12,000×g for 1 minute. Repeat this operation two times. Note: Pre-warm the Eluent or deionized water at 50℃ will generally improve elution efficiency.

Electrophoresis

1. Agalose Gel casting	1) Measure out the appropriate mass of agarose into glass bottle with the appropriate volume of TAE buffer. 2) Microwave until the agarose is fully melted. 3) Pour the agarose solution into the gelbox and let it cool for about 30 minutes, until the gel is solid. 4) Remove comb.
2.Running agalose gel	1) Load 5 μL prepared 1kbp ladder. 2) Mix DNA solution with loading buffer (5x). 3) Load it into agalose gel. 4) Run the gel at ~110 volts for 30 minutes.
3.Visualizing agarose gels	1) Remove gel from gel box. 2) Soak the gel in ethidium bromide solution. 3) Place the gel in Trans-Illuminator and turn on UV light after make sure the door closing. 4) Print the picture. 5) Remove gel and throw in trash.

Restriction Enzyme Digestion

1. Mix the following:

Name	Volume
Sample DNA	4 µg
Restriction Enzyme (EcoRI, XbaI, SpeI, PstI)	0.5µL
10x Buffer	1 µL
ddH2O	up to 10 µL
total	10 µL

2. Let stand for 1 hour at 37 °C.

Ligation

1	Mix the vector DNA and the insert DNA (the vector and the insert at 1 : 5-10).
2	Add 1ul Exnase® II Ligase and 2ul CE II Buffer(5x).
3	Incubate at 16 °C for 1 hour (or at 4 °C for overnight).

Transformation

1	Add 2ul DNA to 150ul chemically conpetent cells on ice (set negative control by using chemically competent E.coli cells without plasmids).
2	Incubate on ice for 30 minutes.
3	Heat shock at 42℃ for exactly 90 seconds.
4	Place samples back on ice for 2 minutes.
5	Operating in the clean bench, add 900ul of LB broth per tube.
6	Incubate at 37℃ for 60 minutes, shaking.
7	Centrifuge at 4000rpm for 1 minute.
8	Operating in the clean bench, discard the supertanant (about 700ul) and resuspend bacteria cells.
9	Use the inoculating loop to load bacteria liquid then streak on the LB plate (+antibiotic selection if necessary).
10	Place plates upside down and incubate at 37℃ overnight.

Fluorometric measurement

1. Add 2ml of the sample to quartz cell.

2. Setting measurement parameter:

EX wavelength	490nm
EM start wavelength	450nm
EM end wavelength	550nm
Scan speed	1200nm/min
EX slit	2.5nm
EM slit	2.5nm
PMT voltage	900V

3. Measure by Fluorospectro photometer model: F-4600 FL Spectrophotometer

HPLC（High Performance Liquid Chromatograpy）

Inject 10μL of the sample into column.HPLC condition will show below：

HPLC	Agilent 1290 Infinity LC
Column	ZORBAX Eclipse® XDB-C18
Total Flow	1mL/min
Pump A	Pump B=H2O
temperature	25℃
Mobile phase	Pump A:Pump B=H2O:MeOH=0.6:0.4

Team:SiCAU-China/Experiments