Team:Chiba/Notebook/protocol


 * E.coli Time Manager  

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Contents

 * 1) Transformation (Using Zymo comp.)
 * 2) DNA_Purification
 * 3) Sigma prep (Plasmid mini prep)
 * 4) Zymo DNA Cleam&Concentrator Kit
 * 5) Agarose gel electrophoresis
 * 6) Digestion
 * 7) PCR
 * 8) Gel extract
 * 9) Dephosphorylation of DNA
 * 10) Ligation

Transformation
Day 1 morning


 * 100 ml SOB medium in 1L or 500 mL flask and sterilize


 * E.coli culture grown in 2 mL of fresh LB medium.

Day 1 night


 * Inoculate preculture (100 μL-1 mL) to sterile SOB medium.Shake culture vigoruoussly at 20-25 °C until OD is 0.4-0.6.

Day 2
 * Transfer the culture to ice 10min.


 * Prepare Wash buffer and Competent buffer by adding 3 mL Dilution buffer to 3 mL of Wash buffer(x2) and to 2.5 mL of Competent buffer(2x), respectively.(on ice)


 * Pellet the cells by centrifugarion at 2500rpm for 6 min.


 * Remove supernatant and genlly resupend the cells in 6 mL ice-cold Wash buffer(1x).


 * Pellet the cells by centrifugarion at 2500rpm for 6 min.


 * Completely remove the supernatant and gently resuspend the cells in 6 mL ice-cold Competent buffer(1x).


 * Aliquot (on ice) 100μL of cell suspension into sterile 1.5 mL microtube and store in deep freezer.

Zymo DNA Cleam&Concentrator Kit

 * Add 2 volume of DNA binding buffer to each volume of DNA sample, Use voltex to mix.


 * Load mixture silica column and place column into a 2 ml collection tube


 * Centrifuge at full speed for 30 sec. Discard the flow-through.


 * Add 200μL of wash buffer and spin 30 sec.


 * Place siliva volumn into a new 1.5 ml tube. Add water directly to the column matrix and spin to elute the DNA.

Agarose gel electrophoresis

 * 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


 * Running agalose gel


 * 1) Load 5 μL prepared 1kbp ladder
 * 2) Mix DNA solution with loading dye(6x) and water
 * 3) Load it into agalose gel
 * 4) Run the gel at ~100 volts for 35 mins.


 * Visualizing agarose gels
 * 1) Remove gel from gel box
 * 2) Soak the gel in ethidium bromide solution
 * 3) Let it 30 min.
 * 4) Place the gel in Trans-Illuminator and turn on UV light after make sure the door closing.
 * 5) Print the picture.
 * 6) Remove gel and throw in trash
 * 7) Wipe down Trans-Illuminator if necessary.

Digestion

 * 1) Mix (in a PCR tube)
 * 2) plasmid DNA
 * 3) buffer
 * 4) Restriction Enzyme
 * 5) NFW
 * 6) Incubate at 37ºC for 3h

PCR

 * Resuspend primer in Nuculease free water to 100 µM

100µL
 * PCR mix
 * 1) DNA template          1µL
 * 2) Fwd primer           10µL (final con. 10 pM)
 * 3) Rev primer           10µL
 * 4) 10x thermo pol buffer 10µL
 * 5) dNTP mix             10µL
 * 6) DNA pol.              1µL
 * 7) dH20                 58µL


 * PCR cycle

Start: 94 °C for 5 min. (melt) cycle:         melt:            1 min. anneal :          30 sec. cycle end: extension:72 °C for 3.5 min. 25 cycles 72 °C for 10 min store: keep at 6 °C forever

Gel extract

 * 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


 * Running agalose gel
 * 1) Load 5 μL prepared 1kbp ladder
 * 2) Mix DNA solution with loading dye(6x) and water
 * 3) Load it into agalose gel
 * 4) Run the gel at ~100 volts for 35 mins.


 * Visualizing agarose gels
 * 1) Remove gel from gel box
 * 2) Soak the gel in ethidium bromide solution
 * 3) Let it 30 min.
 * 4) Place the gel in Trans-Illuminator and turn on UV light after make sure the door closing.
 * 5) Print the picture.
 * 6) Remove gel and throw in trash
 * 7) Wipe down Trans-Illuminator if necessary.


 * Extract
 * 1) Cut the agarose target band
 * 2) The chip of the gel into 2mL ADB buffer
 * 3) Let it in 37 degree 30 min to solve the agarose gel.
 * 4) Purify the DNA with Zymo DNA Cleam&Concentrator Kit

Dephosphorylation of DNA
SAP: Alkaline Phosphatase (Shrimp)


 * Mix
 * 1) DNA fragment 1~10 pmol
 * 2) shrimp Alkaline Phosphatase (1~5 μ l) 1~5 U
 * 3) 10X SAP Buffer 5 μ l
 * 4) Sterilized distilled water up to 50 μ l
 * 5) Incubate at 37°C for 15~30 min.
 * 6) Incubate at 65°C for 15 min. (for inactivation by heat treatment)
 * 7) Purify the DNA with Zymo DNA Cleam&Concentrator Kit

Ligation

 * 1) Mix insert DNA with vector DNA.
 * 2) Add 1u Invitrogen Ligase and Ligase-Buffer(x5).
 * 3) Store RT for 3h.

Time Delay Test

 * 1) Transformed sender and receiver into E coli strains.
 * 2) Inoculated them independently in liquid media. Incubated at 37°C 12h.
 * 3) Inoculated again in Fresh liquid media upto about OD600=2 at 37°C
 * 4) Washed sender and receiver.
 * 5) Mixed them. (Sender:Receiver=1000μL:1000μL)
 * 6) Incubated at 25°C, 30°C　or 37°C.
 * 7) Measured intensity of green fluorescence at regular time intervals.(Fluoroskan AscentR FL＆Fluoroskan AscentR　Thermo ELECTRON CORPORATION)


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