Add 1-5 μl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex
Place the mixture on ice for 2 minutes. Do not mix
Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
Place on ice for 2 minutes. Do not mix.
Pipette 250 μl of room temperature SOC into the mixture. Immediately spread 50-100 μl onto a selection plate and incubate overnight at 37-42°C. NOTE: Selection using antibiotics other than ampicillin may require some outgrowth before plating on selective media. Colonies develop faster at temperatures above 37°C, however some constructs may be unstable at elevated temperatures.
Colony PCR
Conditions：
Gel Recycling
Run electrophoresis separation of the target DNA fragment
Cut off the target DNA fragments, as far as possible the unwanted cut off
The cut of the glue into the 1.5m | plastic centrifuge tube, said the quality of the plastic
Add the same amount of Binding Buffer(XP2), that is, add 0.3m| liquid if 0.3g is weighed
Heat in a 50-60 degree bath for a few minutes until all the glue is melted, stirring to mix.
Put the HiBind DNA Mini Column into the 2ml collection tube
Add the HiBindDNA Mini Column with less than 700ul from the DNA solution in step 5
Centrifuge 60s at 12000rpm at room temperature
Discard the waste liquid and recycle the collection pipe
Repeat steps 7-9 until all samples are transferred to column
Add 300ul Binding Buffer
Centrifuge 60s at 12000rpm at room temperature
Discard the waste liquid and recycle the collection pipe
Add the 700ul SPW Wash Buffer
Centrifuge 60s at 12000rpm at room temperature
Discard the waste liquid and recycle the collection pipe once again in steps 14-16
Centrifuge the empty HiBind DNA Mini Column 1 2000rmp for 2 minutes
The Hibind DNA Mini Column was transferred to 1.5m| plastic centrifuge tube
Add 15-30ul Elution Buffer to the center of the membrane
Let sit at room temperature for 2 minutes
Centrifugation for 12000rmp at room temperature for 1 minute
plasmid extraction from positive clones
using an extraction kit
Decant or aspirate and discard the culture media.Centrifuge at 10,000x g for 1 minute at room temperature
Add 250 uL Solution I/RNase A. Vortex or pipet up and down to mix thoroughly.Complete resuspension of cell pellet is vital for obtaining good yields.
Transfer suspension into a new 15 mL microcentrifuge tube.
Add250 uL Solution IL Invert and gently rotate the tube several times to obtain a dear lysate. A 2-3minute incubation may be necessary.
Add 350uL Solution I Immediately invert several times until a flocculent white precipitate form.
Centrifuge at maximum speed (213,000 x g) for 10 minutes. A compact white pellet will form. Promptly proceed to the next step.
Insert a HiBinde DNA Mini Column into a 2 mL Collection Tube.
Transfer the cleared supernatant from Step 8 by CAREFULLY aspirating it into the HiBind* DNA Mini Column. Be careful not to disturb the pellet and that no cellular debris is transferred to the HiBind" DNA Mini Column.
Centrifuge at maximum speed for 1 minute.
Discard the filtrate and reuse the collection tube.
Add 500 uL HBC Buffer
centrifuge at maximum speed for 1 minute.
Discard the filtrate and reuse collection tube.
Add 700uL DNA Wash buffer.
Centrifuge at maximum speed for 1 minute.
Discard the filtrate and reuse the collection tube.
Centrifuge the empty HiBind" DNA Mini Column for 2 minutes at maximum speed to dry the column matrix.
Transfer the HiBind DNA Mini Column to a clean 1.5 mL microcentrifuge tube.
Add 30100 uL Elution Buffer or sterile deionized water directly to the center of the column membrane.
Let sit at room temperature for 1 minute.
Centrifuge at maximum speed for 1 minute. Note: This represents approximately 70% of bound DNA. An optional second elution will yield any residual DNA, though at a lower concentration
Store DNA at -20degrees.
Purification of recombinant protein with His tag
1. buffer preparation:
Lysis Buffer 1 (under native condition)
0.5mM Tris-HCl
0.5M NaCl
5%(w/v) glycerol
10mM Imidazole
100mg(1mg/ml) lysozyme (Purification of 6xHis-tagged proteins from E.coli)
1% Nonidet P40 (NP40 = Igepal CA-630)
0.25% Tween 20 （or Triton 100）
0.02% NaN3 (Optional)
2 tablets of protease inhibitor cocktail (EDTA free, Recommended)
200 μg(2μg/ml) RNase A (Optional) 1mg (10μg/ml) DNase1 (Optional)
50 mM NaF (Optional)
1mM Na3VO4 (Optional)
+ ddH2O to 100ml, adjust pH to 8.0 using NaOH
Lysis Buffer 2(under denature condition)
50 mM Tris-Cl
8 M Urea (or 6 M Gu-HCl)
10mM Imidazole
0.05% Tween 20
Adjust pH to 8.0 using NaOH
Dialysis/Tev cleavage Buffer
50 mM Tris-Cl, pH8.0
100 mM NaCl (depends on solubility of protein)
2.5% glycerol
0.5mM EDTA
0.5mM DTT or TCEP
Buffer A：
50mM Tris-HCl
0.5M NaCl
5% glycerol
0.05% Tween 20
Adjust pH to 8.0 using high-concentration HCl
Buffer B（washing buffer）：
li50mM Tris-Cl /li
0.5M NaCl 5% glycerol
0.05% Tween 20
500mM imidazole
Adjust pH to 8.0 using high-concentration HCl
Buffer C（imidazole gradient washing buffer）：
Prepare with buffer A and buffer B at different ratio: A 90ml, B 10ml
Sample pretreatment：
fully suspend centrifuge-collected bacteria at the ratio wet bacteria per gram/ 4~5ml Lysis Buffer，and place overnight in -80℃ fridge
thaw the bacteria at 4℃ and suspend the bacteria
break the bacteria in two turns, which contain 5s of ultrasonic breaking, 5s of cooling for each cycle and run 10min per turn, at 400W.
centrifuge at 4℃, 12000rpm for 30min，collect supernatant liquid, and adjust the pH with NaOH to 8.0
take 1ml Ni-NTA agarose gel and place it into 15ml centrifuge tube, then centrifuge at 2000 rpm for 2 min
take the supernatant liquid；add 5ml Lysis Buffer, centrifuge at low speed, then take the supernatant liquid.
add4-5 ml bacteria lysate，and rotate at 4℃ at low speed for 1h
Load the chromatographic column
Wash the chromatographic column with buffer C to wash off protein in the column
