Team:NYCU-Taipei/Experiments

experiments

Check out our experiments and protocols!
Plasmaid Extraction
Preparation
pET-21a in DH5-alpha liquid culture harvest for plasmid extraction
1.  LB: 5ml
2.  Amp(100 mg/ml): 2.5ul
3.  Incubator for 18-19 hour
4.  To pick a single colony, you need to put it on a plate as well. You can also use a tip to do it. Burn the tip a little bit in the alcohol lamp. Use a tip and streak out on the plate, after throw the entire tip into the 15cc tube.
Steps
1.  Harvest
A.  Transfer 1.5ml of cultured bacteria cells to a 1.5 ml Eppendorf.
B.  Centrifuge 14000-16000 x g for 1 min to form cell pellet.
C.  Discard the supernatant.
D.  Repeat the harvest steps using the same Eppendorf. (We use 1 ml one time and repeat 4 times)
2.  Resuspension
A.  Add 200 ul of PD1 buffer(make sure Rnase A was added) to the Eppendorf.
B.  Resuspend the cell pellet completely by vortex or pipette until all traces of the cell pellet have been dissolved. (PD1 is in the 4 degree fridge)
3.  Cell lysis
A.  Add 200 ul of PD2 Buffer to the resuspended sample then mix gently by inverting the tube 10 times. Do not vortex to avoid shearing the genomic DNA.
B.  Let stand at room temperature for at least 2 min. Do not exceed 5 mins.
4.  Neutralization
A.  Add 300 ul of PD3 buffer then mix immediately by inverting the tubes 10 times ( invert it very slowly!).
B.  Centrifuge at 14000-16000 xg for 3 min at room temperature. If using over 5 ml bacteria cells, centrifuge at 16000-20000 xg for 5-8 min.
C.  While waiting, place a PDH Column in a 2 ml collection tube.
5.  DNA binding
A.  Transfer all the supernatant to the PDH column.
B.  Centrifuge at 14000-16000 x g for 30 sec at room temperature then discard the flow-through.
C.  Place the PDH column back in the 2 ml collection tube.
6.  Wash
A.  Add 400 ul of W1 buffer into the PDH column. Centrifuge at 14000-16000 x g for 30 sec.
B.  Discard the flow-through then place the PDH column back in the 2 ml Collection tube.
C.  Add 600 ul of Wash buffer into the PDH column.
D.  Centrifuge at 14000-16000 x g for 30 sec at room temp.
E.  Discard the flow-through then place the PDH column back in the 2ml collection tube.
F.  Centrifuge at 14-16000 x g for 3 min at room temp. to dry the column matrix.
G.  Transfer the dried PDH column to a new 1.5 ml Eppendorf.
7.  Elution
A.  Add 50ul (40 ul for higher concentration) of Elution buffer into the Center of the matrix.
B.  Let stand for at least 2 min to allow Elution Buffer to be completely absorbed.
C.  Centrifuge at 14000-16000 x g 2min at room temp to elute the purified DNA.
Enzyme Digestion
Preparation
Total Volume(20ul)
components amount per reaction (ul)
DNA(1 ug)
10 x buffer 2
Enzyme 0.8-1
Add ddH2O up to 20 uL
components amount per reaction (ul)
DNA(1 ug)
10 x buffer 2
Enzyme1 0.8
Enzyme2 0.8
Add ddH2O up to 20 uL

Steps
1.  Prepare a clean PCR tube to undergo this experiment
2.  Add the components in following order: ddH20, DNA(4°C), 10x buffer(-20°C), enzyme(-20°C)
3.  After adding, pipetting the tube to mix it well
4.  Put the tube into the incubator or water bath(37°C) for 1 hr
Electrophoresis
Preparation
1.  Agarose
A.  Big: 50 ml TAE+ 0.5g agarose(1%) + 1g agarose(2%)
B.  Small: 30 ml TAE+ 0.3g agarose(1%) + 0.6g agarose(2%)
2.  Safety dye: 0.5ul/ 10ml
3.  DNA marker: 5-8 marker
A.  100 bp marker
B.  1 kb marker
4.  Loading dye(6x): ⅕ volume of the sample
Steps
1.  Dissolve the agarose (heat by microwave oven)
2.  Wait to cool a little (you could touch it with your hand to feel the temperature) and add safety dye into it
3.  Pour agarose onto the gel tray
4.  Load sample first, then the marker
5.  Run 70 V, at least 30 min
Gel Extraction
Steps
1.  Gel dissociation
A.  Prepare a 55-60°C water bath. At the same time preheat Elution buffer in the Dry bath(69°C)
B.  Transfer the gel slice to a 1.5 ml Eppendorf
C.  Add 500 ul of gel buffer to the sample then mix by vortex.
D.  Incubate at 55-60°C for 10-15 min to ensure the gel slice is completely dissolved.
E.  During incubation, invert the tube every 2-3 min.
2.  DNA binding
A.  Place a DFH Column in a 2 ml Collection tube.
B.  (Wait to cool down to room temp), spin down and transfer 800ul of the sample mixture to the DFH column.
C.  Centrifuge at 14-16000 x g for 30 sec. Discard the flow-through and put the DFH column back in the 2 ml collection tube. (If the sample mixture is more than 800 ul, repeat the DNA binding step)
3.  Wash
A.  Add 400 ul of W1 buffer into the DFH column.
B.  Centrifuge at 14-16000 x g for 30 sec. Discard the flow-through and place the DFH column back to the collection tube.
C.  Add 600 ul of Wash buffer(make sure ethanol was added) into the DFH column.
D.  Let stand for 1 min at room temp. Centrifuge at 14-16000 x g for 30 sec then discard the flow-through.
E.  Place the DFH column back in the 2 ml collection tube.
F.  Centrifuge at 14-16000 x g for 3 min to dry the matrix.
4.  Elution
A.  Transfer the dried DFH column to a new 1.5 ml Eppendorf.
B.  Add 20-50 ul(25 ul) of pre-heated Elution buffer into the center of the column.
C.  Let stand for at least 2 min (put 5 min).
D.  Centrifuge at 14-16000 x g for 2 min.
Ligation
Preparation
Total Volume(20ul)
components amount per reaction (ul)
T4 DNA ligase buffer 2
Vector DNA (? bp)
Insert DNA (? bp) 2
T4 DNA ligase 1
Add ddH2O up to 20 uL
Calculation
1.  Usually, 1 : 1 or 1 : 3 molar ratio of vector : insert works
Steps
1.  Add the components in following order: water, DNA, T4 DNA ligase buffer, T4 DNA ligase
2.  Use an Eppendorf or PCR tube
3.  Let it stand over 1 hour to let it ligase. (put it in 4 degree if you want to put it overnight)
4.  The next time if you want to transform this product, remember to inactivate (goal : inactivate the ligase, 65 degree 10 min)
5.  Cool down then transform
Transformation
Steps
1.  Prepare 42°C water bath
2.  Prepare the ice with water. Put the sterile Eppendorf on tubes on ice.
3.  Make sure the vector and insert DNA fragment are ready.
4.  Take competent cells out of -80°C and thaw on ice immediately.
5.  Mix 1-5 ul of DNA into 30-35 ul of competent cells. Gently mix by flicking the bottom of the tube.(Do not vortex!)
6.  Incubate the competent cell and DNA mixture on ice for 20-30 mins
7.  Heat shock 42°C water bath for 60 sec.
8.  Put the tubes back on ice for 2 min.
9.  Add 250-1000ul LB(without antibiotic) to the bacteria and grow in 37°C shaking incubator for 45 min
10.  Place the transformation onto LB agar plate with selective marker. (We add 100 ul in every plate)
11.  Put the plate for 14~20 hr in the incubator.
Fibrin Plate
Preparation
(2 petri dishes : total volume of 16ml solution)
1.  Tris-HCl: 20 mmol/L Tris-HCl buffer (pH 8.0)
2.  Agar solution: 0.128g agar in 10 ml Tris-HCl, 50°C
3.  Fibrinogen: 0.032g fibrinogen in 6 ml 0.9% NaCl, 37°C(note: the final concentration comes to 0.8% agar solution, 0.2% fibrinogen)
4.  Thrombin (58.8U/ml): 55ul of thrombin solution in 500 ul of 0.9% NaCl
5.  Filter paper: 《ADVANTEC》NO.1 Qualitative Filter Paper, NO.1
6.  Petri dish: 5.5 cm in diameter
Steps
1.  Prepare a 50°C and 37°C water bath
2.  Prepare the fibrinogen solution and put it in the 37°C water bath for 15 min to dissolve fibrinogen
3.  Dissolve agar and let it cool down to 50°C
4.  Mix both solution and pour into 2 petri dishes (5.5 cm in diameter)
5.  Let stand for 30 min
6.  Add thrombin solution and put a filter paper on top
7.  Let stand for another 30 min to form fibrin clot
8.  Discard the filter paper and punch holes (3 mm of diameter)
9.  Add 10 ul of sample and put it in 37°C incubator for 24 hours to see the lytic circle
Freeze Drying
Preparation
1.  Protective medium: Sucrose (10%), trehalose (5%), Skimmed milk (10%), Ascorbic acid (0.1~0.15%) in PBS solution
2.  0.9% NaCl
3.  PBS: 1x PBS (pH =7.4)
Freeze-dryer
1.  BENCHTOP 2K , VIRTIS
Steps (before freeze)
1.  Bacteria harvest
A.  5 ml LB medium in one 15 ml tube, put it in the incubator to shake for 16 hours
2.  Label 1.5ml Eppendorf
3.  Form cell pellet and load with desired medium
A.  Load 500 ul of bacteria medium in each Eppendorf
B.  Centrifuge for 16000 x g for 1 min to form cell pellet
C.  Discard the supernatant
D.  Load 500 ul in each Eppendorf with desired medium (protective medium or 0.9% NaCl)
4.  Freeze in -80 degree
A.  Put the Eppendorf in -80 degree for at least 5 hours
5.  Transfer to the freeze-dryer
A.  Take out from the fridge and change the lid with holes on to
B.  After changing the lid, put it into the freeze-dryer as fast as possible. (Do not let it melt.)
C.  Freeze-drying condition : 129mTorr (=17.2 Pa), -85.5 degree, time: 24 hours
6.  Count fresh cells before freeze
A.  Label 5 Eppendorf, and put 900 ul PBS solution in each Eppendorf
B.  Take 100 ul of the sample solution and dilute to 10-1~10-5
C.  Use the plate counting method to calculate viable cells
D.  Add 2.5 ul of each diluted sample and spread on the plate
E.  Put in the 37°C incubator and count them after 24 hours
Steps (after freeze)
1.  Take out from the freeze-dryer
A.  Change the lid and use parafilm for longer storage
B.  Put the samples at desired condition and time
C.  Remember to wash the lid with holes and let it dry to reuse.
2.  Rehydration
A.  Add 500 ul of PBS solution to rehydrate the sample
3.  Count fresh cells after freeze
A.  Label 5 Eppendorf, and put 900 ul PBS solution in each Eppendorf
B.  Take 100 ul of the sample solution and dilute 10-1~10-5
C.  Use the plate counting method to calculate viable cells
D.  Add 2.5 ul of each diluted sample and spread on the plate
E.  Put in the 37°C incubator and count them after 24 hours
Competent Cell
DAY-1 Buffer preparation & bottle sterilization
1.  SOB medium (for 250 mL SOB prepared in flask or Duran bottle)
components amount per reaction (gram)
Yeast extract 1.25
Tryptone 5
NaCl 0.15
KCl 0.05
MgSO4 0.6
Add ddH2O up to 250 mL
> Autoclave 121°C, 20 min
> Store at 4°C
2.  CCMB80 transformation buffer (for 1L CCMB80 in flask Duran bottle)
components amount per reaction (gram)
KOAc 0.98 (10mM)
CaCl2 • 2H2O 11.8 (80mM)
MnCl2 • 4H2O 4 (20mM)
MgCl2 • 6H2O 2 (10mM)
Glycerol 100 (10%)
Add ddH2O up to 1L
> Adjust pH to 6.4 with HCl
> Sterile filtered
> Store at 4°C
DAY-2 Preculture
Pick single colony to 3 mL SOB broth, incubate under 37°C, 200 rpm, overnight.
DAY-3
1.  Transfer 100X preculture to new SOB broth inside a flask (5X larger than SOB volume), culture under 37°C, 200 rpm to OD600 = 0.35~0.4
2.  Prepare
A.  A full bucket of ice.
B.  CCMB80 buffer under 4°C.
C.  (optional) Liquid nitrogen in ice bucket.
Every step should be done ON ICE
3.  Following step 1, transfer the cell into 50 mL PP tube. (No more than 25 mL cell in each.) Incubate on ice (covered by ice) for 10 min.
4.  Centrifuge 1600~2500xg, 10 min. under 4°C.
5.  Decant supernatant gently, and stand the tubes in an inverted position for 1 min. to allow the last traces of medium to drain away.
6.  (Per 25 mL cell) resuspend with 10 mL chilled CCMB80 by vortex.
7.  Centrifuge 1600~2500xg, 10 min. under 4°C.
8.  (Per 25 mL cell) resuspend with 1 mL chilled CCMB80 by vortex.
9.  Dispend per 100 μL of cell into a new microcentrifuge tube, and then put it immediately back to ice or liquid nitrogen.
10.  Freeze at -80°C.
Transformation of Competent Cells
1.  Pipet DNA (1 μL of miniprep-grade plasmid DNA, or up to 9 μL of ligation reaction) into an Eppendorf tube and place this on ice. Place an aliquot of competent cells on ice and leave until thawed (~10 minutes).
2.  Add 100 μL of competent cells to the DNA, and incubate for 20-30 minutes on ice.
3.  Heat shock in a water bath at 42°C for 30~60 seconds (if unsure, use 60 seconds) and immediately place on ice.
4.  Add 1 ml of LB medium and shake at 37°C for 30-60 minutes. If you transform miniprep DNA, plate out 100 μL of the cells onto a suitable selective plate. If you transform ligation reactions, spin the cells at 3000 rpm for 5 min. Pour off most of the medium, leaving 100-200 μL in the tube. Resuspend the cells in this remaining medium and plate everything onto suitable selective plates.
PCR
Preparation
Total Volume(20ul)
components Sample (ul) Control (ul)
10X KOD buffer 2.5 2.5
Mg2+ 1 1
DNA template 50 ng (if using gDNA template, increase the amount of the template)
dNTP (2mM) 2.5 2.5
primer pair 1.5 1.5
KOD polymerase 0.5 0.5
Add ddH2O up to 25 uL

set up PCR machine
1.  HOLD: 94°C, 1 min.
2.  Cycling (*30)
3.  Denaturing - 94°C, 20 sec
4.  Annealing - 55°C, 30 sec. (or other appropriate annealing temperature)
5.  Extending - 68°C, 1 min. (adjust the time length by the size of the predicted product)
6.  HOLD: 68°C, 10 min.
7.  HOLD: 20°C, 15 min.
8.  (if we need touchdown, touchdown 10 cycle, then choose an appropriate annealing temperature for 25 cycle)
RFP intensity measurement
Steps
1.  Making L-arabinose solution
A.  Weigh 6 g L-arabinose and add ddH2O to form a 20 ml solution(2M).
B.  Draw the L-arabinose solution into a 20cc (or 50 cc) syringe.
C.  Place a 0.22um filter onto the syringe. Force the solution through the syringe into a sterile 50 ml centrifuge tube.
2.  Inoculate a liquid culture of the provided bacteria
A.  Prepare a sterile culture tube. Label properly.
B.  Put an inoculating loop into the flame until it is red hot, and wait for a few seconds to allow it to cool.
C.  Use the sterile inoculating loop to pick a bit of the bacteria on the plate and stir into the culture tube.
D.  Recap the tube and incubate it in a shaking incubator overnight.
3.  Test the concentration of cells by ODE testing. Record the OD600 value and the estimated concentration of cells
See Appendix 1.
4.  Inoculate liquid cultures with different concentrations of L-arabinose
A.  Take five 15 ml centrifuge tubes or culture tubes, and label them as 0.2M, 0.1M, 0.05M, 0.025M, 0M(negative control), respectively.
B.  Transfer 5 ml sterile LB medium into each tube.
C.  Add 2 μl of 1000x (100 mg/ml) ampicillin stock solution into each tube. Mix thoroughly. (Final concentration: 100 μg/ml).
D.  Add 222 ul 2M L-arabinose solution into the 0.2M centrifuge centrifuge tube.
E.  Add 105 ul 2M L-arabinose solution into the 0.1M centrifuge centrifuge tube.
F.  Add 50 ul 2M L-arabinose solution into the 0.05M centrifuge centrifuge tube.
G.  Add 25 ul 2M L-arabinose solution into the 0.025M centrifuge centrifuge tube.
H.  Add 5 ul of the bacterial liquid culture into each tube. Mix thoroughly.
I.  Test the concentration of cells of each culture by ODE testing according to the Appendix 1.
J.  Measure RFP intensity of each culture according to Appendix 2.
K.  Put the tubes into a shaking incubator , temperature at 37 °C and shaking speed at 200 RPM.
L.  Test for the concentration of cells and RFP intensity for each tubes according to Appendices 1 & 2 after 12, 24,36 ,and 48hr of incubation.
5.  Retrieve the data from the computer, correct for the measurements for the OD600 and subtract the autofluorescence from the control sample of all the other samples
Appendices
1.  Testing the concentration of cells by ODE testing
A.  setting parameter in the plate reader
i.  Measurement: Absorbance
ii.  Set Temperature: 37°C
iii.  Shake: Medium, 0:30 (MM:SS)
iv.  Multiple Reader per well: 3x3(square)
A.  Set the absorption wavelength to 600 nm and make sure the measuring unit is set to Abs (or A).
B.  Transfer 40ul LB blank and 160 ul PBS buffer into 96 well plate(with clear bottom and flat white sides).
C.  Set the absorption wavelength to 600 nm and make sure the measuring unit is set to Abs (or A).
D.  Transfer 40ul LB blank and 160 ul PBS buffer into 96 well plate(with clear bottom and flat white sides).
E.  Set the absorption wavelength to 600 nm and make sure the measuring unit is set to Abs (or A).
2.  Test the RFP intensity of each culture
A.  Load 160 µl PBS buffer in one well of the 96-well plate (with clear bottom and black sides) and add 40 µl of LB blank .
B.  Load 160 µl PBS buffer in the wells of the 96-well plate (with clear bottom and black sides) and add 40 µl of bacteria culture .
C.  setting parameter in the plate reader
i.  Measurement:Fluorescence Intensity
ii.  Set Temperature: 37°C
iii.  Shake: Medium, 6:00 (MM:SS)
iv.  Excitation: 555,Emission: 585
v.  Optics: Bottom,Gain: 100
vi.  Multiple Reader per well: 3x3(square)
vii.  Read Speed: Normal, Delay: 100 msec, Measurements/Data Point: 10
D.  Start the plate reader machine.
6x Histag purification
Growth of expression cultures
1.  Inoculate 10 ml of LB medium containing the appropriate antibiotics with a fresh bacterial colony harboring the expression plasmid. Grow at 37°C overnight.
2.  Dilute the non-induced overnight culture 1:50 (e.g., inoculate 100 ml LB medium with 2 ml overnight culture) with fresh LB medium containing the appropriate antibiotics. Grow at 37°C with vigorous shaking until the OD600 reaches 0.6~0.8.
3.  Add IPTG to a final concentration of 1 mM and grow the culture at 37°C with vigorous shaking for 4 hours. (In Nattokinase expression, we use 20°C for 16 hr). IPTG (Isopropyl β-d-1-thiogalactopyranoside): MW 238.30.
4.  Harvest the cells by centrifugation at 8000 rpm for 15 min.
Preparation
(for native condition purification) 1.  Lysis Buffer (NPI-10): 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0.
2.  Wash Buffer (NPI-20): 50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0.
3.  Elute Buffer (NPI-500): 50 mM NaH2PO4, 300 mM NaCl, 500 mM imidazole, pH 8.0.
4.  Benzonase® Endonuclease 25 U/μl.
5.  Lysozyme stock solution 10 mg/ml in water. Sterilize by filtration and store in aliquots at –20°C.
Purification
(native condition) 1.  Resuspend a pellet derived from 5 ml cell culture volume in 630 μl Lysis Buffer (NPI-10). Add 70 μl Lysozyme Stock Solution (10 mg/ml) and add 3 Units/ml culture volume Benzonase® Nuclease.
A.  If the tagged protein does not bind under these conditions, the amount of imidazole should be reduced to 1-5 mM.
2.  Incubate on ice for 15–30 min.
3.  Centrifuge lysate at 12,000 x g for 15–30 min at 4°C. Collect supernatant.
A.  Save 20 ul of the cleared lysate for SDS-PAGE analysis.
4.  Equilibrate the Ni-NTA spin column with 600 ul Buffer NPI-10. Centrifuge for 2 min at 890 x g.
A.  The spin column should be centrifuged with an open lid to ensure that the centrifugation step is completed after 2 min.
5.  Load up to 600 ul of the cleared lysate containing 6xHis-tagged protein onto the pre-equilibrated Ni-NTA column. Centrifuge for 5 min at 270 x g.
A.  To ensure efficient binding it is important not to exceed 270 x g with centrifugation.
B.  For very concentrated cell lysate, it may be necessary to extend the centrifugation time to 5 or 10 min at 270 x g.
C.  Save the flow-through for analysis by SDS-PAGE to check binding efficiency.
6.  Wash the Ni-NTA spin column twice with 600 ul Buffer NPI-20. Centrifuge for 2 min at 890 x g.
A.  For very low expression levels or highly concentrated lysates, 3 wash steps may be required to achieve high purity.
B.  Save the flow-through (wash fractions) for analysis by SDS-PAGE to check the stringency of the wash conditions.
7.  Elute the protein twice with 300 ul Buffer NPI-500. Centrifuge for 2 min at 890 x g, and collect the elute.
A.  If higher protein concentrations are desired, do not combine the eluates or, alternatively, elute 100-200 ul aliquots.
SDS page for protein expression
Sampling
1.  For liquid culture: incubate bacteria in LB with corresponding antibiotic to OD600 = 0.4~0.6. Take 1 mL to microcentrifuge tube and centrifuge for 16,000 g for 1 minute.
2.  For protein induction after liquid culture: Take 1 mL of inducted bacterial medium and measure OD600. Centrifuge for 16,000 g for 1 minute in microcentrifuge tube.
3.  To make the band in SDS page clear, samples have to be dilution. For example,
if OD600 = 1.537, add 153.7 λ of ddH2O for dilution. Diluted sample 30 λ + 10 λ 4X dye, cook 100°C for 10 minutes
SDS-PAGE
1.  Prepare gel
A.  Set up the glass plates and put them onto the green clamp.
i.  Nudge the set to the end of the loading stand and fix it with the clamp above.
B.  Loading with ddH2O first for 10 secs to check if the gel stands have been firmly set. Pour out ddH2O and dry up glass plates with paper towel.
C.  Prepare separating gel in a tube.
D.  Prepare separating gel in a tube.
SDS-Page Separating Gel (for 1mm page)
6% 8% 10% 12% 15% 18% 20%
ddH2O (ml) 2.6 2.3 1.9 1.6 1.1 0.6 0.7
1.5M Tris pH8.8 (ml) 1.3
10% SDS (ml) 0.05
10% APS (ml) 0.05
30% Acrylamide 29:1 (ml) 1 1.3 1.7 2 2.5 3 3.33
TEMED (ml) 0.002
Total volume (ml) 5.02
E.  Pipetting and stir with p1000, suck up about 4.5 ml solution and load between the glasses to 2 cm below the green stick.
i.  Load 70% EtOH between the glasses to full.
ii.  Wait for 20 mins for separating gel to dried up.
iii.  * Notice that APS and TEMED will solidify the gel. Add them the LAST.
F.  Prepare 5% stacking gel in a tube.
SDS-Page Stacking Gel (5%)
2ml 4ml
ddH2O (ml) 1.4 2.8
1.5M Tris pH6.8 (ml) 0.25 0.5
10% SDS (ml) 0.02 0.04
10% APS (ml) 0.02 0.04
30% Acrylamide 29:1 (ml) 0.33 0.66
TEMED (ml) 0.002 0.004
Total volume (ml) 2.022 4.044
G.  Pipetting and stir with p1000, suck up about 2 ml solution and load between the glasses to full.
i.  Put on the comb (Don’t make bubbles!)
ii.  Wait for 20 mins.
H.  Unload the glasses with gel (comb remained), store in water inside a box under 4°C if needed.
2.  Running SDS page
A.  Prepare 10X tris-Glycine SDS running buffer (700 ml).
i.  70 ml SDS stock buffer + 630 ml ddH₂0, mix well.
B.  Set up the SDS box (with glasses on both sides).
i.  Small glass facing inside.
ii.  Plug into the bottom with a tilt.
iii.  Push the glasses upwards to the block.
iiii.  Push the handles inwards to fix the glasses.
C.  Load samples: prestained protein marker 3~5 λ, samples 5~10 λ.
D.  Running: stacking gel 70~90V for 30 minutes (standard), separating gel 120V 1.5 for hours.
3.  Staining and destaining of SDS page
A.  Remove the glass plates from the gel carefully in the water.
B.  Stain the gel with coomassie blue for 20 minutes on the shaker (60 rpm).
C.  Destain with destaining solution (50% ddH₂0, 40% Methanol, 10% Acetic acid) or in water overnight on the shaker (60 rpm).