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.
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
Agalose Gel casting
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
Running agalose gel
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.
Visualizing agarose gels
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
Mix (in a PCR tube)
plasmid DNA
buffer
Restriction Enzyme
NFW
Incubate at 37ºC for 3h
PCR
Resuspend primer in Nuculease free water to 100 µM
PCR mix
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
----------------------------
100µL
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
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
Running agalose gel
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.
Visualizing agarose gels
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.
Extract
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.
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