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Contents
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Protocols
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 ℃ 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.
DNA Purification
Sigma prep
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
- Measure out the appropriate mass of agarose into glass bottle with the appropriate volume of TAE buffer
- Microwave until the agarose is fully melted
- Pour the agarose solution into the gelbox and let it cool for about 30 minutes, until the gel is solid
- Remove comb
- Load 5 μL prepared 1kbp ladder
- Mix DNA solution with loading dye(6x) and water
- Load it into agalose gel
- Run the gel at ~100 volts for 35 mins.
- Remove gel from gel box
- Soak the gel in ethidium bromide solution
- Let it 30 min.
- Place the gel in Trans-Illuminator and turn on UV light after make sure the door closing.
- Print the picture.
- Remove gel and throw in trash
- Wipe down Trans-Illuminator if necessary.
Digestion
PCR
- Resuspend primer in Nuculease free water to 100 µM
- DNA template 1µL
- Fwd primer 10µL (final con. 10 pM)
- Rev primer 10µL
- 10x thermo pol buffer 10µL
- dNTP mix 10µL
- DNA pol. 1µL
- dH20 58µL
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100µL
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
READ:Agarose_gel_electrophoresis
- Run the Digested DNA solution
- Cut the agarose target band
- The chip of the gel into 2mL ADB buffer
- Let it in 37 degree 30 min to solve the agarose gel.
- Purify the DNA with Zymo DNA Cleam&Concentrator Kit
READ:Zymo_DNA_Cleam&Concentrator_Kit
Dephosphorylation of DNA
SAP: Alkaline Phosphatase (Shrimp)
DNA fragment 1~10 pmol
Shrimp Alkaline Phosphatase (1~5 μ l) 1~5 U
10X SAP Buffer 5 μ l
Sterilized distilled water up to 50 μ l
- Incubate at 37℃ for 15~30 min.
- Incubate at 65℃ for 15 min. (for inactivation by heat treatment)
- Purify the DNA with Zymo DNA Cleam&Concentrator Kit
READ:Zymo cleam
Phosphorylation of DNA
Ligation
Time Delay Test
Time Delay Test
- Transformed sender and receiver into E coli strains.
- Inoculated them independently in liquid media. Incubated at 37℃ 12h.
- Inoculated again in Fresh liquid media upto about OD600=2 at 37℃
- Washed sender and receiver.
- Mixed them. (Sender:Receiver=1000μL:1000μL)
- Incubated at 25°C, 30°C or 37°C.
- Measured intensity of green fluorescence at regular time intervals.(Fluoroskan AscentR FL＆Fluoroskan AscentR Thermo ELECTRON CORPORATION)
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