1. Prepare Solutions
2. Gel Electrophoresis
(1) Load protein and molecular weight marker
(2) Add running buffer
(3) Electrify: set the voltage at 80V before the sample reach the dividing line between stacking gel and resolving gel,switch to 130V until the blue stain reach the buttom line
3. Blotting
I. Transfer (1)Carefully cut the gel
(2)Assemble the transfer cassette
(3)Install the cassette and electrify to transfer the protein to PVDF
II. Chemiluminescene (1) Blocking: 5% whole milk incubate for 1h
(2) Incubate with diluted primary antibody
(3) Wash membrane: TBST 15 mins *4
(4) Incubate with diluted secondary antibody
(5) Wash membrane: TBST 15 mins *4
(6) Add Chemiluminescene substrate, incubate in dark for 5 mins
(7) Exposure
Immunoprecipitation(IP)
→ Reagents :
Lysis buffer
Tris-HCl: 50 mM, pH 7.4
NP-40: 1%
Na-deoxycholate: 0.5%
NaCl: 150 mM
COMPLETE protease inhibitors (Roche)
→ Protocol :
1. Use 1 x 10 cm dish of near confluent cells for 1 ml of lysis buffer.
2. Wash cells in 2 ml ice cold PBS.
3. Lyse cells with 1 ml of 1x lysis buffer containing protease inhibitor cocktail (PIC). Wash cells off of plate
using a pipette or cell scraper.
4. Centrifuge at 4℃ for 15 mins at 15000 rpm to pellet insoluble material.
5. Carefully remove supernatant to a fresh tube.
6. Add 1 μg of primary antibody and incubate with gentle mixing for 2-16 hours at 4℃.
7. Wash 20 ml of Protein G-sepharose in 1ml of lysis buffer
8. Resuspend to 100 μl and add to lysate containing antibody.
9. Incubate 1 hour with gentle mixing at 4℃.
10. Wash complexes 3 times in lysis buffer + PIC at 4℃.
11. Boil in 50 μl SDS sample buffer prior to loading on gel
12. Run on SDS-PAGE and immunoblot.
Electrical transfection
→ Reagents :
DNA: 0.2 - 2.0µg/µl purified plasmid DNA
Pipette glass: glass capillary tubing, boroscilicate, standard wall with filament (World Precision Instruments
Inc.)
Electrode tip diameter will depend on the application.
→ Protocol :
Micropipettes : A micropipette and Picospritzer are used to pressure injected DNA into the brain ventricle. The shape
and size of the pipette tip is not critical, but it must be sharp enough to easily pierce the tissue, and large
enough to quickly deliver the DNA. We use the Picospritzer II to deliver 75-125nl DNA solution directly into
the tadpole brain ventricle. The same pipette is used for multiple animals.
DNA solution : We test a wide range of plasmid concentrations (using Clontech pEGFP) and find that concentrations
ranging between 0.2 - 2.0µg/µl yield comparable numbers of fluorescent cells, with similar intensity of GFP fluorescence.
DNA can be diluted in dH2O, buffered saline, or 2mM CaCl2. The DNA solution was colored with 0.01% fast green
as a visual aid for filling the brain ventricle. For co-electroporation of two plasmids, we mix plasmids in a
ratio of 1:1. This typically gives a co-transfection rate of 70% ±10% (determined for the simultaneous electroporation
of of pEGFP and pDsRed).
Set-up:A dissecting microscope with good optics is imperative. The micro-manipulators are placed either side of
the stage. One manipulator connected to the Picospritzer holds the micropipette, and the other one holds
the platinum electrodes connected to the capacitor and stimulator.
Procedure : The anesthetized tadpole is placed on a moistened kimwipe on the center of the microscope stage.
The micropipette containing DNA is inserted into the ventricle of the tadpole brain, and the DNA is injected into the ventricle under pressure. For widespread electroporation, DNA is injected to fill the entire brain ventricle.
For targeted electroporation of a specific brain region, a concentrated bolus of DNA should be injected as close
as possible to the region of interest. The micropipette is removed, and the platinum electrodes are immediately
lowered to contact the tadpole's skin, spanning the brain region of interest (see Fig. 2). 2-7 voltage pulses
are delivered (depending on desired level of transfection). Effervescent bubbles are produced at the electrode
tips where they contact the skin. The level of effervescence is a good indicator of whether you have achieved
electroporation vs electrocution. There should be numerous small bubbles along the electrode tips.
If the bubbles are large and bubbling over, the voltage will be too high and the animal will die. Another visual
cue is the amount of shock the tadpole displays. The tadpole eyes usually flick in response to the electroporation.
If the whole body jolts, the voltage must be too large. After electroporation, the tadpole is quickly returned to
rearing solution, where it usually recovers within 10 minutes.
The DNA constructs can be targeted to just one side of the brain, or if possible, the whole brain can be transfected.
This is achieved by regulating the voltage polarity. If only one side of the brain is to be transfected, the
polarity setting on the stimulator is set so the negatively charged DNA moves towards the positive electrode.
If both sides of the brain are to be transfected, the polarity must be switched while the voltage pulses are
being delivered.
Stimulation parameters : Depending on the number of transfection cells desired, 2 - 7 pulses of 30 - 50V with
an exponential decay of 70 ms is optimal. To transfect fewer cells, we should reduce the numbers of pulses.
Detecting transfected cells : Transfected cells expressing GFP are detected by standard fluorescence microscopy.
Trouble shooting : We occasionally see some bleeding in the brain ventricle 24 hrs after electroporation. This
usually clears up by 48 hrs. Propidium iodide staining indicated that electroporation does not cause an increase
in cell death. For good charge conduction: - Ensure that the specimen remains moist - The platinum electrodes
must be cleaned regularly.
Plasmid extraction
→ Reagents :
Solution I
50 mM glucose
25 mM Tris-Cl (pH 8.0)
10 mM EDTA (pH 8.0)
Autoclave, and store at room temperature.
Solution II
0.2N NaOH
1% SDS
Store at room temperature in a plastic bottle. Don't autoclave.
Solution III
5M potassium acetate 60 ml
glacial acetic acid 11.5 ml
Distilled water 28.5 ml
Store at room temperature. This solution can be autoclaved. However, we usually use this solution without autclaving.
→ Protocol :
1.Shake E. coli harboring plasmid at 37 C overnight in 50 ml of TB containing appropriate antibiotics. (when using
ampicillin, addition of the antibiotics to 100-200 ug/ml rather than usual 50 ug/ml may improve the yield of
plasmids.)
2. Spin the bacterial culture at 6,000 rpm for 10 mins at 4℃. Discard the supernatant.
3. (0ptional) Suspend the cell in about 20 ml of deionized water. Spin again. Discard the supernatant. Remove
all of the liquid supernatant using pipetman.
4. Resuspend the pellet in 5 ml of Solution I.
5. Add 10 ml of Solution II. Mix well by inverting the bottle more than 10 times. The solution should be clear
after mixing Solution II.
6. Add 7.5 ml of Solution III. Mix well as above.
7. Spin the lysate at 10,000 rpm for 10 mins at 4℃.
8. Transfer the supernatant into a new bottle. Add about 15 ml of isopropanol, mix well, and store the bottle
for 10 mins at room temperature.
9. Spin at 10,000 rpm for 10 mins at 4℃. Discard the supernatant. Remove all of the fluid using pipetman.
10. Dissolve the pellet in 600 ul of TE. Transfer the solution into a microfuge tube.
11. Add 200 ul of 8M LiCl. Mix well, and then spin the solution at 14,000 rpm for 5 mins at 4℃.
12. Transfer the supernatant containing plasmid DNA to a new microfuge tube. Add 600 ul of isopropanol. Mix
well, and then spin the solution at 14,000 rpm for 5 mins at 4℃.
13. Discard the supernatant. Rinse the pellet and the wall of the tube with 500 ul of cold 70% ethanol. Discard
the fluid.
14. Add to the pellet 400 ul of TE containing DNase-free RNase A (20 ug/ml). Incubate the tube for 30 mins at
37 C.
15. After 30 mins, carefully check the content of the tube. If nucleic acid pellet is visible at the bottom of
the tube, vortex well to dissolve the pellet. Incubate the tube at 37°C for further 30 mins.
16. Add 240 ul of 2M NaCl, 20% PEG8000. (PEG6000 supplied from Japanese suppliers is essentially equivalent
to PEG8000, and works well.)
17. Spin at 14,000 rpm for 5 mins. Discard the supernatant. Rinse the pellet with 300 ul of cold 70% ethanol.
Discard the fluid. Dissolve the pellet in 400 ul of TE. (Optional: Repeat PEG precipitation once more. This is
recommended for preparing dephosphorylated linear vector since trace amount of short RNA in the vector preparation
may interfere with the dephosphorylation reaction.)
18. Extract the plasmid solution with chloroform to remove PEG. Take aquaous phase. Extract the aquaous phase
with phenol. Take aquaous phase. (Optional: Repeat phenol extraction until no interphase is visible. This is
recommended for preparing the template for in vitro transcription.)
Extract the aquaous phase with chloroform or ethylether to remove trace amount of phenol dissolved in the solution.
Take aquaous phase (or remove organic phase).
19. Add 0.1 volume of 3 M sodium acetate and 3 volume of ethanol into the plasmid solution. Spin at 14,000 rpm
for 5 mins at 4℃. Discard the supernatant. Rinse the pellet with 200 - 500 ul of 70% ethanol.
20. Store the open tube on the bench until the visible traces of ethanol have evaporated.
21. Dissolve the DNA pellet in 200-500 ul of TE. Typically 300-800 ug of plasmid should be obtained if the plasmid
have pUC-based replication origin.
Lentivirus packaging
→ Production of Lentiviral Vectors: Packaging of the Vectors :
1.Maintain 293T cells in complete culture medium in a 37°C incubator with 5% CO2. Twenty-four hours before transfection,
plate exponentially growing 293T cells in 100-mm tissue culture dishes at 4 x 106 cells/plate. Cell density should be approximately 80% confluent for transfection.
2.Prepare 1 ml of calcium phosphate-DNA suspension for each 100-mm plate of cells as follows:
3.Set up two sterile tubes for transfection of one plate. Label the tubes 1 and 2.
4.Add 0.5 ml of 2x HBS to Tube 1.
5.Add TE 79/10 to Tube 2. The volume of TE 79/10 = 440 µl minus the volume of the DNA solution.
6.Add 15 µg of the transfer vector containing the transgene, 15 µg of pCgp, 5 µg of pCMV-rev,
and 5 µg of pCMV-G to Tube 2 and mix.
7.Add 60 µl of 2 M CaCl2 solution to Tube 2 and mix gently.
8.Transfer the contents from Tube 2 to Tube 1 dropwise with gentle mixing.
9.Allow the suspension to sit for 30 minutes at room temperature.
→ Mix the precipitate well by pipetting or vortexing
1.Add 1 ml of the suspension to a 100-mm plate containing cells. Add the suspension slowly,
dropwise while gently swirling the medium in the plate. Return the plates to the 37°C incubator and
leave the precipitate for 4 hours.
2.Replace the old medium with 6 ml of fresh culture medium. Add 60 µl of 0.6 M sodium butyrate. Return to the incubator.
3.After 48 hours of culture, collect the supernatant and freeze it at -80℃ or proceed to the concentration step.
→ Concentration of the Vectors
1.Centrifuge the supernatant (freshly collected or thawed from the freezer) at 900g for 10 minutes to
remove any cell debris in the supernatant.
2.Filter the supernatant through a 0.2-µm syringe filter.
3.Transfer the supernatant to autoclaved polyallomer tubes. Concentrate the supernatant by ultracentrifugation
for 1.5 hours at 4℃ in a Beckman SW 28 swinging bucket rotor at 24,500 rpm.
4.Remove the supernatant and resuspend the pellet in an appropriate amount of culture medium, e.g.,
300 µl for 30 ml of original supernatant if a 100-fold concentration is desired.
5.Divide the concentrated vector into 10-50-µl aliquots and store at -80℃ until use.
→ Titration of the Vectors
1.Seed 5 x 104 HT1080 cells/well in a 12-well plate in complete medium and culture overnight in a 37°C incubator with 5% CO2.
2.Add serial diluted vector stock and 4 µl/ml polybrene to the cultured cells. Continue culture for 48 hours.
3.Trypsinize the cells. Following centrifugation, remove the supernatant and resuspend the pellet in 300 µl of 3.7% formaldehyde in PBS.
4.Determine the percentage of EGFP-positive cells by FACS analysis.
5.The titer will be represented as transduction units (TUs) per milliliter concentrated vector (TU/ml).
Lentivirus transfection
→ Transduction of Lentiviral Vectors to Target Cells
1.Seed exponentially growing cells at 2 x 105 cells/well in 1 ml of culture medium into a 24-well plate.
2.Add various amounts of concentrated vector stock depending on cell type.
3.Add 4 µg/ml polybrene. Return the cells to a 37°C incubator.
4.After overnight incubation, centrifuge the cells, discard the supernatant, and resuspend
the cells with fresh culture medium. Return the cells to culture.
5.Determine transduction efficiency by FACS analysis 48 hours after transduction (see Troubleshooting).
6.Purify CD34+ hematopoietic stem cells from umbilical cord blood or bone marrow using anti-CD34
antibody-coupled magnetic beads following the manufacturer's protocol.
7.Forty-eight hours before transduction, culture the CD34+ cells in CD34+ transduction medium.
8.Coat a 48-well nontissue-culture-treated plate with 0.2 ml of 25 µg/ml RetroNectin (~5 µg/cm2) for 2 hours at room temperature
9.Remove RetroNectin and then add 0.2 ml of 2% BSA in PBS for blocking. Store the plate for 30 minutes at room temperature.
10.After washing the wells with PBS, adjust the lentiviral vector stock to the appropriate moi (range 5-40)
with plain IMDM medium to 200-µl volume and load it into the well of the coated plate.
11.After incubation for 2 hours at 37°C, remove the vector supernatant and then wash the well with PBS.
12.Add the prestimulated CD34+ cells to the well at 1 x 105 cells/well in 0.2 ml of growth medium and
return the cells to the 37°C incubator.
13.After overnight culture, centrifuge the cells, resuspend the cell pellet in 1 ml of culture medium,
and transfer the cells to a 24-well plate. Return the cells to the incubator.
14.Determine transduction efficiency by FACS analysis 6 days after transduction.
1. Components: total volume 20μL
10×buffer 2μL dNTPs mixture (10mmol) 0.4μL MgCl 2 1.2μL Taq 0.2μL Sense primer (10mM) 0.5μL Antisense primer (10mM) 0.5μL ddH O 13.1μL DNA 1μL
2. Cycling Conditions:
Step 1: 95℃, 5mins
Step 2: 95℃, 30s
Step 3: 95℃, 30s
Step 4: 95℃, 30s (fluorescence detection)
Step2-Step4, 40 cycles (variable, can be up to 45 cycles)
3. Data Analysis: The Comparative Ct Method (ΔΔCT Method)
CT---cycles when the reaction reach the threshold, the relative expression level of each miRNA
compares to endogenous Control can be described as 2-ΔCT, (ΔCT= CT sample- CT endogenous control).
GAPDH, a housekeeping gene, is usually used as endogenous Control for mRNA.
RNA extraction using TRIzol/TRI
→ Reagents:
TRIzol or TRI reagent
0.8 M sodium citrate / 1.2 M NaCl
isopropanol (2-propanol)
chloroform
75% EtOH in DEPC H2O
RNase free water (filtered or DEPC)
→ Protocol:
1.cell lysis
Cell lysis only takes a few minutes per well, but tissue homogenisation can take 10-20 minutes per sample depending on how tough the tissue is.
(1) (PBS wash)
(2) add trizol (cell lysis)
1ml / 3.5 cm diameter well (6-well)
5ml / 75 ml bottle
(3) homogenise by pipetting several times (mechanic lysis)
alternative for tubes: vortex 1 min
alternative for tissue: grind 1 g tissue in liquid nitrogen in a motar and pestle, put tissue into plastic screw-cap centrifuge tube + 15 ml TRIzol reagent, incubate samples for 5 mins at room temp or 60° C (scaled up as needed)
(4) (5 mins at RT for complete dissociation of nucleoprotein complexes)
RNA is stable in trizol which deactivates RNases. You can take a break at this point keeping the sample
in trizol for a short time or freezing it for a longer one.
2.phase separation
15-45 mins depending on number of samples and whether an additional chloroform wash is necessary
(1) add chloroform (1/5 volume of trizol; e.g. 0.2ml to 1ml)
(2) shake for 15 sec (Eccles protocol: do not vortex)
(3) incubate 2-5 mins at RT
(4) spin max. 12000g, 5-15 mins, 2-8°C
if centrifugation hasn't been sufficient the DNA-containing interphase will be cloud-like and poorly compacted
If supernatant appears turbid an additional chloroform cleaning step can be inserted here.
(5) transfer aqueous upper phase into new tube
Take care not to aspirate the DNA-containing white interface. This quickly happens and will lead to DNA contamination in your RNA prep.
TRIZOL phases after chloroform addition
TOP - colourless aqueous phase (RNA) - 60% TRIZOL volume
MIDDLE - interphase (DNA)
BOTTOM - red (organic) phenol-chloroform phase (proteins & lipids)
3.RNA precipitation and wash
20-40 mins depending on number of samples
(1) add isopropanol (70% of aqueous phase or 1/2 trizol volume)
(2) 0.8 M sodium citrate or 1.2 M NaCl can be added
(3) (incubate 10 mins at RT)
(4) spin max g, 10-15 mins, 4℃
(5) remove supernatant
(6) (alternative RNA precipitation - RNeasy from Qiagen) better than alcohol precipitation for smaller
amounts of RNA (less risk of losing a miniscule nucleic acid pellet); also reduces risk of organic solvent contamination
similar kits to RNeasy: MinElute kit, or Affymetrix sample clean-up
4.RNA wash
15-30 mins( depending on number of samples)
(1) wash pellet 70% EtOH (add & vortex briefly)
70% ethanol prepared with RNase-free water
some prefer to wash the pellot more than once with 70% ethanol
(2) spin max g, 2-10 mins, 4℃
(3) air-dry pellet for 5-10 mins Do not overdry the pellet or you won''t be able to redissolve it.
optional add RNase inhibitor
(4) incubate at 55-60 C° for 10 mins if hard to redissolve
(5) transfer to eppendorf tube
(6) spin 4℃, 5 mins (to pellet undissolved material)
5.redissolving of RNA
(1) dissolve pellet in 50-100 µl filtered or DEPC H2O (note: DEPC inhibits RT reaction)
(2) alternatively, 0.5% SDS, pipetting up and down, heat to 55-60°C for 10 mins
