Team:NJU China/Protocol

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Protocol

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Subculturing Cells

Plasmind DNA Transfection

Subculturing Cells Ready to passage when cell confluence to 80%, and the procedure is carried out in the super clean bench.
1.Remove present culture media.
2.Wash cells with PBS to remove the residual media.
3. Add 2 mL of trypsin, and let the cells sit for 2-5 minutes at 37℃ until the cells separated from the culture dish. Note:It may be necessary to bang the culture flasks on the hood counter to remove any “sticky” cells from the flask surface.
4. Immediately after the 2 minutes, add room temp DMEM media (10%FBS) to inactivate the trypsin, and passing the cell several times through a pipette.
5. Transfer cell suspension to 15 ml tube, centrifuge at 1000rpm for 5 minutes. Remove supernatant.
6. Add 5 mL of PBS, mix immediately by pipetting. centrifuge at 1000rpm for 5 minutes. Remove supernatant. Add DMEM to resuspend the cells
7. Add cell culture medium DMEM to a new flask(or dish).
8. Add cell suspension into the new flask in a certain ratio (according to the result of counting, see Cell Counting), and mix it well.
9. Place culture flasks back into the 5% CO2 incubator and check daily
Cell Counting
1. Add 5 μL of cell suspension into 45 μL PBS
2. Put 10 μL of the mixture onto the counting cell slide.
3. Count the cells in the four corner boxes and the middle box.Count border cells on only two of the borders (top and left or bottom and right etc.)
5. The number of cells in the suspension = number of cells * 104/4 * dilution ratio per milliliter.

Plasmind DNA Transfection Use the following procedure to transfect DNA into mammalian cells in 24-well format. For other formats, see Scaling Up or Down Transfections. All amounts and volumes are given on a per well basis. Prepare complexes using a DNA(μg) to Lipofecctamin TM 2000(μl) ratio of 1:2 to 1:3 for most cell lines. Transfect cells at high cell density for high efficiency, high expression levels, and to minimize cytotoxicity, Optimization may be necessary(see Optimizing Plasmid DNA Transfection).
1. Adherent cells: One day before transfection, plate 0.5-2105 cells in 500μl of growth medium without antibiotics so that cells will be 90%-95% confluent at the time of transfection. Suspension cells: Just prior to preparing complexes, plate 4-8105 cells in 500μl of growth medium without antibiotics.
2. For each transfection sample, prepare complexes as follows:
a. Dilute DNA in 50μl of Opti-MEM
b. Mix Lipofectamine TM2000 gently before use, then dilute the appropriate amount in 50μl of opti-MEM ⅠMedium, Incubate for 5 minutes at room temperature. Note: Proceed to Step c Within 25 mintues.
c. After the 5 minute incubation, combine the diluted DNA with diluted Lipofetcamine TM 2000 (total volume=100μl) Mix gently and incubate for 20 mintues at room temperature (solution may appear cloudy). Note: Complexes are stable for 6 hours at room temperature.
3. Add the 100μl of complexes to each well containing cells and medium. Mix gently by rocking the plate back and forth.
4. Incubate cells at 37℃ in a CO2 incubator for 18-48 hours prior to testing for transgene expression. Medium may be changed after 4-6 hours.
5. For stable cell lines: Passage cells at a 1:10 (or higher dilution) into fresh growth medium 24 hours after transfection. Add Selective medium (if desired) the following day. Scaling Up or Down Transfections To transfect cells in different tissue culture formats, vary the amount of Lipofectamine TM 2000, nucleic acid, cells, and medium used in proportion to the relative surface area, as shown in the table. With automated, high-throughput systems a complexing volume of 50μl is recommended for transfections in96-well plates. Note: You may perform rapid 96-well plate transfections by plating cells directly into the transfection mix. Prepare complexes in the plate and directly add cells at twice the cell density as in the basic protocol in a 100μl volume. Cells will adhere as usual in the presence of complexes.

1Surface areas may vary depending on the manufacturer.
2Volumes of dilution medium in Step 2a&2b of DNA or RNAi transfection protocols.

Optimizing Plasmid DNA Transfection
To obtain the highest transfection efficiency and low cytotoxicity, optimize trans fection conditions by varying cell density as well as DNA and LipofectamineTM 2000 concentrations. Make sure that cells are greater than 90% confluent and vary DNA(μg): LipofectamineTM 2000(μl) ratio of 1:0.5 to 1:5.

RNA Extraction

RT-PCR

RNA Extraction A:TRIzol Reagent (Invitrogen) _Instructions For RNA Isolation
Caution: When working with TRIzol Reagent use gloves and eye protection (shield, safety goggles). Avoid contact with skin or clothing. Use in a chemical fume hood. Aviod breathing vapor.
Unless otherwise stated, the procedure is carried out at 15 to 30°C, and reagent are at 15 to 30°C.
Reagent required, but not supplied:
Chloroform
Isopropyl alcohol
75% Ethanol (in DEPC-treated water)
RNase-free water or 0.5% SDS solution [ To prepare RNase-free water, draw water into RNase-free galss bottles. Add diethylpyrocarbonate (DEPC) to 0.01% (v/v). Let stand overnight and autoclave. The SDS solution must be prepared using EDPC-treated, autoclaved water.]
1. HOMOGENIZATION (see notes 1-3)
　　　a. Tissues
　　　Homogenize tissue samples in 1 ml of TRIzol Reagent per 50-100 mg of tissue using a galss-Teflon or power homogenizer (Polytron, or Tekmar’s TISSUMIZER or equivalent). The sample volume should not exceed 10% of the volume of TRIzol Reagent used for homogenization.
　　　b. Cells Grown in Monolayer
　　　Lyse cells directly in a culture dish by adding 1 ml of TRIzol Reagent to a 3.5 cm diameter dish, and passing the cell lysate several times through a pipette. The amount of TRIzol Reagent added is based on the area of the culture dish (1 ml per 10 cm2) and not on the number of cell present. An insufficient amount of TRIzol Reagent may result in contamination of the isolated RNA with DNA.
　　　c. Cells Grown in Suspension
　　　Pellet cells by centrifugation. Lyse cells in TRIzol Reagent by repetitive pipetting. Use 1 ml of the reagent per 5-10×106 of animal, plant or yeast cells, or per 1×107 bacterial cells. Washing cells before addition of TRIzol Reagent should be avoided as this increases the possibility of mRNA degradation. Disruption of some yeast and bacterial cells may require the use of a homogenizer.
　OPTIONAL: An additional isolation step may be required for samples with high content of proteins, fat, polysaccharides or extracellular material such as muscles, fat tissue, and tuberous parts of plants. Following homogenization, remove insoluble material from the homogenate by centrifugation at 12000×g for 10 minutes at 2 to 8°C. The resulting pellet contains extracellular membranes, polysaccharides, and high molecular weight DNA, while the supernatant contains RNA. In samples from fat tissue, an excess of fat collects as a top layer which should be removed. In each case, transfer the cleared homogenate solution to a fresh tube and proceed with chloroform addition and phase separation as described.
2. PHASE SEPARATION
　Incubate the homogenized samples for 5 minutes at 15 to 30°C to permit the complete dissociation of nucleoprotein complexes. Add 0.2 ml of chloroform per 1 ml of TRIZOL Reagent. Cap sample tubes securely. Shake tubes vigorously by hand for 15 seconds and incubate them at 15 to 30°C for 2 to 3 minutes. Centrifuge the samples at no more than 12,000×g for 15 minutes at 2 to 8°C. Following centrifugation, the mixture separates into a lower red, phenol-chloroform phase, an interphase, and a colorless upper aqueous phase. RNA remains exclusively in the aqueous phase. The volume of the aqueous phase is about 60% of the volume of TRIZOL Reagent used for homogenization.
3. RNA PRECIPITATION
　Transfer the aqueous phase to a fresh tube, and save the organic phase if isolation of DNA or protein is desired. Precipitate the RNA from the aqueous phase by mixing with isopropyl alcohol. Use 0.5 ml of isopropyl alcohol per 1 ml of TRIZOL Reagent used for the initial homogenization. Incubate samples at 15 to 30°C for 10 minutes and centrifuge at no more than 12,000×g for 10 minutes at 2 to 8°C. The RNA precipitate, often invisible before centrifugation, forms a gel-like pellet on the side and bottom of the tube.
4. RNA WASH
　Remove the supernatant. Wash the RNA pellet once with 75% ethanol, adding at least 1 ml of 75% ethanol per 1 ml of TRIZOL Reagent used for the initial homogenization. Mix the sample by vortexing and centrifuge at no more than 7,500 × g for 5 minutes at 2 to 8°C.
5. REDISSOLVING THE RNA
　At the end of the procedure, briefly dry the RNA pellet (air-dry or vacuum-dry for 5-10 minutes). Do not dry the RNA by centrifugation under vacuum. It is important not to let the RNA pellet dry completely as this will greatly decrease its solubility. Partially dissolved RNA samples have an A260/280 ratio < 1.6. Dissolve RNA in RNase-free water or 0.5% SDS solution by passing the solution a few times through a pipette tip, and incubating for 10 minutes at 55 to 60°C. (Avoid SDS when RNA will be used in subsequent enzymatic reactions.) RNA can also be redissolved in 100% formamide (deionized) and stored at -70°C (5). RNA Isolation Notes:
1. Isolation of RNA from small quantities of tissue (1 to 10 mg) or Cell (102 to 104) Samples: Add 800 μl of TRIZOL to the tissue or cells. Following sample lysis, add chloroform and proceed with the phase separation as described in step 2. Prior to precipitating the RNA with isopropyl alcohol, add 5-10 μg RNase-free glycogen (Cat. No 10814) as carrier to the aqueous phase. To reduce viscosity, shear the genomic DNA with 2 passes through a 26 gauge needle prior to chloroform addition. The glycogen remains in the aqueous phase and is co-precipitated with the RNA. It does not inhibit first-strand synthesis at concentrations up to 4 mg/ml and does not inhibit PCR.
2. After homogenization and before addition of chloroform, samples can be stored at -60 to -70°C for at least one month. The RNA precipitate (step 4, RNA WASH) can be stored in 75% ethanol at 2 to 8°C for at least one week, or at least one year at –5 to -20°C.
3. Table-top centrifuges that can attain a maximum of 2,600 × g are suitable for use in these protocols if the centrifugation time is increased to 30-60 minutes in steps 2 and 3.
B:TRIzol LS Reagent_RNA Isolation Procedure
Always use the appropriate precautions to avoid RNase contamination when preparing and handling RNA.
RNA pricipitation
1. (Optional) When precipitating RNA from small sample quantities(<106 cells or <10 mg tissue), add 5-10μg of RNase-free glycogen as a carrier to the aqueous phase. Note: Glycogen is co-precipitated with the RNA, but does not inhibit first-strand synthesis at concentrations ≤4mg/mL, and does not inhibit PCR.
2. Add 0.5mL of 100% isopropanol to the aqueous phase, per of 0.75 mL TRIzol LS Reagent used for homogenization.
3. Incubate at room temperature for 10 minutes.
4. Centrifuge at 12,000×g for 10 minutes at 4°C.
Note: The RNA is often invisible prior to centrifugation, and forms a gel-like pellet on the side and bottom of the tube.
5. Proceed to RNA wash and resuspension.
RNA wash and resuspension
1. Remove the supernatant from the tube, leaving only the RNA pellet.
2. Wash the RNA pellet, with 1mL of 75% ethanol per 0.75 mL of TRIzol LS Reagent used for the initial homogenization. Vortex the sample to mix.
3. Centrifuge the sample at 7500×g for 5 minutes at 4°C, and discard the supernatant.
4. Vacuum or air dry the RNA pellet for 5-10 minutes. Do not dry the pellet by vacuum centrifuge.
Note: Do not allow the RNA to dry completely, because the pellet can lose solubility. Partially dissolved RNA samples have an A260/280 ratio <1.6.
5. Resuspend the RNA pellet in RNase-free water or 0.5% SDS solution (20-50μL) by passing the solution up and down several times through a pipette tip. Note: Do not dissolve the RNA in 0.5% SDS if it is to be used in subsequent enzymatic reactions.
6. Incubate in a water bath or heat block set at 55-60°C for 10-15 minutes.
7. Proceed to downstream application, or store at -70°C.

RT-PCR 1. According to the manufacturer’s instruction, use tubes to compound these reagent that listed below. All procedures were carried out on ice.

2. Set the PCR as requested below. Then, start to reverse-transcribe the target RNA
16℃ 30minutes
42℃ 30minutes 1 cycle
85℃ 5minutes
4℃ preserved
Note: qRT-PCR was carried out using a Taqman miRNA PCR kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. Briefly, 1 μl (approximate 1μg/μl) of total RNA was reverse-transcribed to cDNA using AMV reverse transcriptase (TaKaRa, Dalian, China) and the stem-loop RT primers (Applied Biosystems). Real-time PCR was performed using SYBR (Applied Biosystems) on the Applied Biosystems 7300 Sequence Detection System (Applied Biosystems). All reactions, including the no-template controls, were run in triplicate. After the reactions, the CT values were determined using the fixed threshold settings.
Absolute Quantification: To calculate the absolute expression levels of the target siRNAs, a series of synthetic siRNA oligonucleotides (dissolved in water) of known concentrations (from 1 fM to 105 fM) were also reverse-transcribed and amplified. The absolute amount of each siRNA was then calculated by referring to the standard curve. Relative Quantification: To determine the relatively increasing or decreasing level of the target mRNA, U6 and β-actin were also reverse-transcribed and amplified, used for qRT-PCR analysis. The change of target mRNA was then determined by the comparison between U6 and mRNA, or between β-actin and mRNA.

qPCR

Plasmids transformation

qPCR 1. According to the manufacturer’s instruction, use tubes to compound these reagent that listed below. All procedures were carried out on ice.

2. Set the PCR as requested below. Then, start to reverse-transcribe the target RNA

Plasmids transformation
1. Obtain a competent cell aliquot from the -80°C freezer, keep the tubes on ice.
2. Add 40μL of ice cold H2O to each aliquot to make a 80 μL solution.
3. Separate the 80 uL solution into 2 separate tubes to make two 40uL tubes of solution.
4. Add 1μL of a Gibson Product into each tube
5. Using the Electroporator:
a. Set the electroporator to 1250 V. and press "time constant".
b. Obtain a chilled cuvette, and pipet all 40 uL of a sample into the center.
c. Place the cuvette into the electroporator and press "Pluse" twice.
d. Immediately remove the cuvette and rescue the sample in 300-500 mL of LB.
e. Pipet the sample into a labeled microcentrifuge tube, and record the time constant. (The time constant should have a value greater than 2.5).
f. Repeat this process for the other sample. After the addition of LB, transfer the sample into another labeled microcentrifuge tube.
6. Incubate both samples for about 1 hour at 37°C.
7. After incubation, combine both samples (make sure each sample has a comparable time constant) and follow standard plating procedures.

Plasmids extraction

Plasmids extraction with small quantity

Plasmids extraction Preparation
1.Column equilibration: place a Spin Column CP5 into 50 ml collection tube (supplied in the kit) and add 2.5 ml Buffer BL to Spin Column CP5. Centrifuge for 2 min at 8,000 rpm (~8,228 × g). Discard the flow-throw, and place Spin Column CP5 into the same collection tube.
2.Composition of solution:
a)Solution I: 50 mM Glucose / 25 mM Tris-Cl / 10 mM EDTA，pH 8.0
b)Solution II: 0.2 N NaOH / 1% SDS；
c)Solution III: 3 M potassium acetate/ 2 M acetic acid

Protocol
1.Inoculate 1L LB/ampicillin (50μg/mL) medium placed in a 5 liter culture flask with E.coli carrying desired plasmid and grow at 37/C with agitation for 12-16 hr.
2.Pellet up to 1-2L bacteria in appropriate vessels by centrifugation at 4,000 rpm for 15 min at 4°C.
3.Decant or aspirate medium and discard. Add 18 mL Solution I. Resuspend cells completely by vortexing or pipetting up and down. Complete resuspension of cell pellet is vital for obtaining good yield.
4.Add 40 mL Solution II, gently mix by inverting and rotating tube several times. Incubate 10 minutes at room temperature.
5.Add 30 mL chilled Solution III, cover, and gently mix by inverting tube several times until a flocculent white precipitate forms. Incubate on ice for 10 minutes.
6.Centrifuge at 7000rpm for 15 minutes at 4oC to pellet the cellular debris and genomic DNA.
7.Transfer the supernatant into CS1 filter.
8.Measure the volume of the flow-through and add 0.6 volume of the isopropyl alcohol. Mix by inverting the bottle 15-25 times. Incubate on ice for 10 minutes.
9.Centrifuge at 7000rpm for 15min. Carefully decant the supernatant without disturbing the pellet..
10.Resuspend pelleted bacterial cells in 8 ml Buffer P1. Note: Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of the pellet.
11.Add 8 ml Buffer P2 and mix thoroughly by inverting the tube 6-8 times, and incubating at room temperature for 5 min.
Note: Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min.
12.Add 8 ml Buffer P4 and mix immediately and thoroughly by inverting the tube 6-8 times, incubate at room temperature for 10 min. The solution should become cloudy.
　　Note: To avoid localized precipitation, mix the solution thoroughly, immediately after addition of Buffer P4. Centrifuge for 10 min at 8,000 rpm (~8,228 × g). A compact white pellet will form.
13.Note: If use more than 100 ml bacterial culture, prolong centrifugal time to 20-30 min.
14.Transfer the lysate into the Filtration Column CS. Gently insert the plunger into the Filtration Column CS and filter the cell lysate into a new 50 ml tube (not supplied in the kit).
15.Add 0.3 volume isopropanol to the cleared lysate, sealed the received tube, and mix completely and then transfer all solution to the Spin Column CP5. Centrifuge for 2 min at 8,000 rpm (~8,228 × g).
　　Note: If the volume of isopropanol–lysate mixture is larger than the capacity of the column, it can be loaded the column two times.
16. Discard the flow-through and place the Spin Column CB5 back into the same collection tube.
17. Add 10 ml Buffer PW to the column and centrifuge at 8,000 rpm (~8,228 × g) for 2 min. Discard the flow-through and place the Spin Columns CB5 back into the same collection tube.
18. Repeat step17.
19. Add 3ml 96%-100% ethanol to the Spin Column CP5 (put the CP5 in a collection tube). Centrifuge for 2 min at 8,000 rpm (~8,228 × g).
20. Discard the flow-through and centrifuge at 8,000 rpm (~8,228 × g) for an additional 5 min for removing residual ethanol.
　　Note: Residual ethanol will influence the subsequent enzymatic reaction and sequence. Place the column with cap open in air for several minutes to dry the membrane.
21. To elute DNA, place the column in a clean 50 ml collection tube(supplied in the kit) and add 1-2 ml Buffer TB to the center of the membrane and incubate 5 min at room temperature, centrifuge at 8,000 rpm (~8,228 × g) for 2 min.
　　Note: Repeat step 14 to increase plasmid callback efficiency.If the volume of eluted buffer is less than 1 ml, it may affect recovery efficiency. The pH value of eluted buffer will have some influence in eluting. Buffer TB or distilled water (pH 7.0-8.5) is suggested to elute plasmid DNA. For long-term storage of DNA, eluting in Buffer TB and storing at –20°C is recommended, since DNA stored in water is subject to acid hydrolysis.

siRNA screening (eliminated 308 siRNA, 467 siRNA and 516 siRNA seem to have good effect) 19th: We cultured 293t cells in 12-well plates.
20th: We transfected 293t cells.
21st: We collected cells and preserved in Trizol.
22nd: We extracted RNA from cells and did RT-PCR.
24th: We did qPCR.

July

2013

WEEK 8 19th-24th, June

siRNA screening (failed) 8th: We cultured Hep G2 cells in 2 12-well plates for 24-hour cell collection and 48-hour cell collection.
9th: We transfected cells with empty Lipo, HbsAg plasmids, HbsAg plasmids and 467 siRNA plasmids, HbsAg plasmids and 516 siRNA plasmids, respectively.
10th: We collected cells 24 hours after transfection, and preserved them in Trizol.
11th: We collected 48 hours after transfection, and then extracted RNA from 2 groups of cells (24 hours and 48 hours).
12th: We did RT-PCR and qPCR with all RNA samples.
13th: We analyzed the data.

July

2013

WEEK 8 19th-24th, June

siRNA screening (failed) 8th: We cultured Hep G2 cells in 2 12-well plates for 24-hour cell collection and 48-hour cell collection.
9th: We transfected cells with empty Lipo, HbsAg plasmids, HbsAg plasmids and 467 siRNA plasmids, HbsAg plasmids and 516 siRNA plasmids, respectively.
10th: We collected cells 24 hours after transfection, and preserved them in Trizol.
11th: We collected 48 hours after transfection, and then extracted RNA from 2 groups of cells (24 hours and 48 hours).
12th: We did RT-PCR and qPCR with all RNA samples.
13th: We analyzed the data.
Absolute quantification of exosomes (failed) 5th: We cultured 293t cells.
6th: Cells we cultured on 5th July were contaminated by bacteria.
7th: We subcultured another cell line of 293t cells.
9th: We cultured 293t cells in 6 flasks.
10th:We transferred 293T cells with plasmids.
11th: We collected exosomes 24 hours after transfection.
12th: A part of cells died, failed to collect 48-hour exosomes.
13th: We examined protein concentration of 24-hour exosomes, started to extract RNA from 24h-cells and 24h-exosomes and preserved the rough RNA extract solution with isopropyl alcohol at 4℃ overnight.
14th：We continued finish RNA extraction, then did RT-PCR and qPCR.
15th: We analyzed data.
Luciferase Assay 11th: Construction of plasmids: obtain vector and segment.
12th: We combined vector and segment with T4 ligase, then transformed the recombined plasmids into E.coli DH5α competent cells and spread them on solid LB culture plates by streak plate method.
13th: No single bacterial colony was found.
14th: No single bacterial colony was found again, redid previous steps(obtain the target segments, combined vector and segment, and then transferred it into E.coli )
15th: Single bacterial colonies were found. We picked single colonies and transferred them into liquid LB culture medium with ampicillin, and shaken overnight at 37℃.
16th: The transformed E.coli cells failed to reproduce in LB culture medium.
Others 5th: We thawed 293t cells.
6th: We subcultured HepG2 cells.
7th: E.coli cells containing HBSag overexpressed plasmids were transferred respectively into LB culture medium with ampicillin, and shaken overnight at 37℃.
8th: We extracted HBSag overexpressed plasmids from E.coli cells.
11th: We thawed 293t cells and subcultured HepG2 cells. E.coli cells containing GFP plasmids were transferred into LB culture medium with ampicillin, and shaken overnight at 37℃.
12th: We extracted GFP plasmids from E.coli cells.
14th: We thawed HepG2 cells and 293T cells.
15th: We thawed 293T cells.

WEEK 9 AND WEEK 10 16th -21th,22th-28th,July

Others 16th: We subcultured 293T cells. E.coli DH5α competent cells containing GFP plasmids and RVG plasmids were transferred respectively into LB culture medium with ampicillin, and shaken overnight at 37℃.
17th: We found that wrong antibiotics were added into the culture medium of RVG plasmid-containing E.coli cells (it ought to be kanamycin). So we did transferred E.coli cells containing RVG plasmids into LB culture medium with kanamycin), and shaken overnight at 37℃.
18th: We preserved the RVG and GFP strains at -80℃ and extracted plasmids from the culture medium (RVG,GFP, 1L medium). And cryopreserved 293T cells.
Absolute quantification of exosomes (succeeded) 18th: We cultured 293t cells.
19th: We transfected 293T cells with 467 and 516 plasmids(lipo2,4672,5162).
20th: We changed the transfection medium with culture medium and then collected culture medium containing exosomes 24 hours after transection (pre-centrifuge).
21th: We collected culture medium containing exosomes 48 hours after transfection (pre-centrifuge) and soaked.
centrifuge tubes which then be used in ultracentrifugation overnight in DEPC solution(1:1000).
22th: We separated exosomes by ultracentrifugation(110000g).
23th: We examined protein concentration of exosomes collected by ultracentrifugation, and then extracted RNA from these exosomes and cells which we used to produce exosomes and preserved the RNA extracts in isopropyl alcohol over night.
24th：We continued to extract RNA, examined total RNA concentration of exosomes and cells, then RT-PCR RNA 467 and 516.
25th: Q-PCR the cDNA of RNA 467 and 516.
26th: We analyzed data and found that the standard curve cannot be used.
27th: We redid the Q-PCR and analyzed data and this time we succeeded
Examination whether RVG-lamp2b exosomes will target to dendritic cells or not (failed) 18th: We cultured 293t cells.
19th: We transfected 9 flasks of 293T cells with RVG plasmids(lipo3,RVG3,non-related plasmids3).
20th: We changed the 19th transfection medium with cell culture medium 6 hours after transfection and transfected another 9 flasks of 293T cells as we did with the former 9 flasks.
21th: We collected 48-hour cell culture medium transfected on 19th July and pre-centrifuged to remove cell debris and organelles. We soaked centrifuge tubes which then be used in ultracentrifugation overnight in DEPC solution(1:1000).
22th: We collected 48-hour culture transfected on 20th July and pre-centrifuged to remove cell debris and organelles. Meanwhile, we watched green fluorescent in cells. Then we separated exosomes by ultracentrifugation (110000g).
23th: We examined protein concentration of exosomes collected on 22th July, diluted exosome solution from 500μL to 1000μL and filtered the exosome solution then injected exosome solution into the C57 mice. We took 3μL exosomes solution to extract RNA, and preserved RNA extracts in isopropyl alcohol over night.
24th：We continued to extract RNA, and examined total RNA concentration of exosomes and cells which we used to produce these exosomes, then RT –PCR.
25th: We injected exosome solution into the mice for the second time. Q-PCR the cDNA got from RT-PCR on 24th July.
26th: We analyzed data and found the standard curve cannot be used.
27th: We anatomized C57 mice and collected the brain, heart, liver, spleen, lung, kidney and blood of them and redid RT-PCR and Q-PCR with the RNA extracted on 24th July and analyzed data.
28th: We extracted RNA from brain and serum collected on 27th July

August

2013

WEEK 11 29th July-4th, August

Luciferase Assay (failed) 28th: We transfected 293T cells with plasmids in 24-well format.
29th: We did luciferase assay with 293T cells transfected on 28th July.
3th: We redid luciferase assay.
Collection of exosomes containing 467 siRNA & 516 siRNA 29th: We transfected 4 flasks of 293T cells with 467 and 516 plasmids (467 plasmid2, 516 plasmid2).
31th: We collected 48-hour cell culture medium and pre-centrifuged it to remove cell debris and organelles and stored the medium at 4℃. We soaked centrifuge tubes which then be used in ultracentrifugation overnight in DEPC solution(1:1000).
1th: We separated exosomes by ultracentrifugation and stored the exosome solution at 4℃.
Pre-experiment to examine whether anti 214 RNA is high in brain or not (failed) 30th: E.coli DH5α Competent Cells containing anti 214 plasmids were transferred into LB culture medium with spectinomycin, and shaken overnight at 37℃.
31th: We extracted anti 214 plasmid, subcultured 293T cells and transfected 293T cells with anti-214 plasmid. 1th: We collected 24-hour cells and preserved it in Trizol.
2th: We extracted RNA, RT-qPCR anti 214 RNA.
3th: We did agarose gel electrophoresis. Redid this pre-experiment.
Others: 1th: We subcultured and cryopreserved 293T cells.
3th: E.coli DH5α Competent Cells transformed HER2 plasmids were transferred into LB culture medium with spectinomycin, and shaken overnight at 37℃.
4th: We extracted HER2 plasmids and examined DNA concentration.

WEEK 12 5th-12th, August

Pre-experiment to examine whether the expression level of anti 214/her2/467/516 RNA is high in brain or not (success) 7th: We subcultured 293T cells in 12-well format and transfected 293T cells with anti 214/her2/467/516 plasmids and changed the cell culture medium 6 hours after transfection.
8th: We extracted RNA from cells. RNA stored in -80℃.
10th: RT-PCR.
11th: Q-PCR and analyzed data.
12th: Redid the Q-PCR and analyzed data.
Coculture of HepG2 cells transformed HBsAg plasmids with exosomes containing 467 plasmid (failed) 7th: We subcultured HepG2 cells in 12-well format.
8th: We transfected HepG2 cells with HBsAg plasmids and added 467 exosomes into culture medium after 18 hours.
10th: We extracted RNA from HepG2 cells cocultured on 8th August and RT-PCR the RNA of HBsAg.
11th: We Q-PCR the cDNA we got on 10th August and analyzed data.
12th: We redid the Q-PCR and analyzed data.
Others: 7th: We examines protein concentration of exosomes (467, 516 and empty)

WEEK 13 12th-18th, August

Collection of exosomes containing empty/516/516+RVG plasmids 12th: We subcultured four 225cm2 flasks of 293T cells.
13th: We subcultured sixteen 225cm2 flasks of 293T cells.
15th: We transfected 293T cells with plasmids (empty/516/516+RVG) and changed the culture media 6 hours after transfection.
17th: We collected culture media. Meanwhile we soaked centrifuge tubes which then be used in ultracentrifugation overnight in DEPC solution(1:1000).
18th: We separated exosomes by ultracentrifugation and stored the exosome solution at 4℃.
19th: We examined protein concentration of exosomes collected on 18th August(empty, 516,516+RVG).
Pre-experiments for exosomes co-culture experiment. 15th: We subcultured HepG2 cells into a 12-well format.
16th: We transfected HepG2 cells with HBsAg plasmids and cluture medium was contaminated. So we subcultured HepG2 cells into a 12-well format again.
17th: We transfected HepG2 cells with HBsAg plasmids.
18th: We added 467 exosomes into culture media 18 hours after transfection and then collected HepG2 cells 12 hours after transfection and preserved them in Trizol.
19th: We extracted RNA from HepG2 cells. RT-PCR and Q-OCR the it.
Others: 15th: We did RT-PCR, Q-PCR to obtain standard curves of 467 siRNA and 516 siRNA.
17th: We subcultured 293T cells.

WEEK 14 19th-25th, August

RVG targeting experiment 19th:We practised mouse tail intravenous injection.
20th: We practised mouse tail intravenous injection.
21th: We dosed 9 mice with exosomes (empty3, 516,3516+RVG*3) by tail intravenous injection.
22th: We anatomized C57 mice and collected the brain, heart, liver, spleen, lung, kidney and blood of them and preserved these tissues at -80℃.
23th: We extracted RNA from brain and serum we collected on 22th August. Then We did RT-PCR with the RNA.
24th: We did Q-PCR with the cDNA we got on 23th August.
Absolute quantification of exosomes (empty, 516,516+RVG) 20th: We extracted RNA from exosomes (empty, 516,516+RVG) and did RT-PCR with the RNA.
Others: 20th: We subcultured HepG2 cells.
21th: We subcultured 293T cells.

September

2013

WEEK 15 26th August-1th September

Redoing the experiment on 23th August 31th: We extract RNA from brain and serum we collected on 22th August.
1th: We did RT-PCR, Q-PCR with the RNA we extracted on 31th August.

WEEK 16-17 2th-11th, September

Exosomes co-culture experiment 31th: We subcultured HepG2 cells into 12-well format.
1th: We transfected HepG2 cells with HBsAg overexpression plasmids and changed transfection media with culture medium 6 hours after transfection.
2th: We added 467 exosomes to the culture medium 18 hours after transfection.
3th: We extracted RNA from HepG2 cells we transfected on 1th September and used the RNA to do RT-PCR and Q-PCR.
4th: We redid RT-PCR and Q-PCR on 3th September.
Exosomes collection 4th: We transfected 293T cells with 467 plasmids.
5th: We soaked centrifuge tubes which then be used in ultracentrifugation overnight in DEPC solution(1:1000).
6th: We collected culture medium and separated exosomes by ultracentrifugation and stored the exosome solution at 4℃.
Construction of standardized plasmid 1th: We transformated E.coli DH5α Competent Cells with pSB1C3 plasmids and spread these cells on the surface of solid LB medium added chloromycetin.
2th: We transferred single colony into fluid LB culture medium with chloromycetin, and shaken the medium overnight at 37℃.
3th: We extracted plasmids from culture medium shaken on 2th September and examined the DNA concentration of plasmids. Then we digested plasmids with XbaⅠ enzyme and SpeⅠenzyme. We did agarose gel electrophoriesis to test the effect of enzyme digestion. To get more pSB1C3 plasmids, we shaken E.coli DH5α Competent Cells containing pSB1C3 plasmids in fluid LB culture medium overnight at 37℃.
4th: We extracted plasmids from fluid LB culture medium shaken on 4th September.
5th: We digested a lot of pSB1C3 plasmids we extracted on 3th September with XbaⅠ enzyme and SpeⅠenzyme and did agarose gel electrophoriesis to recycle carrier segment by gel extraction kit.
6th: We digested a lot of pSB1C3 plasmids we extracted on 4th September with XbaⅠ enzyme and SpeⅠenzyme and did agarose gel electrophoriesis to recycle carrier segment by gel extraction kit. We linked carrier segment we got on 5th with 467 double-strand segment and 516 double-strand segment by T4 DNA ligase.
7th: We transformated E.coli DH5α Competent Cells with recombined plasmids we got on 6th September and spread these cells on the surface of solid LB medium added chloromycetin.
8th: We transferred single colony of recombination plasmids-containing E.coli cells into fluid LB culture medium with chloromycetin, and shaken the medium overnight at 37℃.
9th: We extracted plasmids from the LB culture medium we shaken on 8th September and sent the recombined plasmids sample to GenScript for sequencing.
10th:
11th: We got result of sequencing from GenScript. We constructed standardized 467-plasmid successfully.

@@ Line 565: / Line 565: @@
                      <div class="ss-row">
                      <div class="ss-left">
-                     <h2 id="March">Subculturing Cells</h2>
+                     <h4 id="March">Subculturing Cells</h4>
                      </div>
                      <div class="ss-right">
@@ Line 623: / Line 623: @@
      		    <div class="ss-row">
                      <div class="ss-left">
-                     <h2 id="April">RNA Extraction</h2>
+                     <h4 id="April">RNA Extraction</h4>
                      </div>
                      <div class="ss-right">
-                     <h2>4. RT-PCR</h2>
+                     <h4>RT-PCR</h4>
                      </div>
                      </div>
@@ Line 708: / Line 708: @@
 		    <div class="ss-row">
                      <div class="ss-left">
-                     <h2 id="May">qPCR</h2>
+                     <h4 id="May">qPCR</h4>
                      </div>
                      <div class="ss-right">
@@ Line 730: / Line 730: @@
 <div class="ss-right">
 <h3>
-<a>siRNA screening (Failed), examination whether siRNA are capsulated into exosomes (Success)</a>
+<a>Plasmids transformation</a>
 <span>
-th: We extracted plasmids of 3 kinds of siRNA.</br>
+</br>1. Obtain a competent cell aliquot from the -80°C freezer, keep the tubes on ice.
-th: We cultured 293t cells in eight D10 dishes and a 12-well plate.</br>
+</br>2. Add 40μL of ice cold H2O to each aliquot to make a 80 μL solution.
-th: We extracted 2 kinds of over-expression plasmids and examined the concentration of it. Then we transfect these plasmids into 293t cells respectively.</br>
+</br>3. Separate the 80 uL solution into 2 separate tubes to make two 40uL tubes of solution.
-th: We collected cells and exosomes from 8 D10 dish, and cells from 12-well plate.</br>
+</br>4. Add 1μL of a Gibson Product into each tube
-th: We extracted RNA from cells and exosomes.</br>
+</br>5. Using the Electroporator:
-th: We did RT-PCR and qPCR.</br>
+</br>a. Set the electroporator to 1250 V. and press "time constant".
+</br>b. Obtain a chilled cuvette, and pipet all 40 uL of a sample into the center.
+</br>c. Place the cuvette into the electroporator and press "Pluse" twice.
+</br>d. Immediately remove the cuvette and rescue the sample in 300-500 mL of LB.
+</br>e. Pipet the sample into a labeled microcentrifuge tube, and record the time constant. (The time constant should have a value greater than 2.5).
+</br>f. Repeat this process for the other sample. After the addition of LB, transfer the sample into another labeled microcentrifuge tube.
+</br>6. Incubate both samples for about 1 hour at 37°C.
+</br>7. After incubation, combine both samples (make sure each sample has a comparable time constant) and follow standard plating procedures.
 </span>
 </h3>
@@ Line 744: / Line 751: @@
-<div class="ss-row ss-medium">
-<div class="ss-left">
-<h3>
-<a>WEEK 5</a>
-<span>15th-18th, May</span>
-</h3>
-</div>
-<div class="ss-right">
-<h3>
-<a>siRNA screening (Failed)</a>
-<span>
-th: We cultured the 293t cells in a 12-well plate.</br>
-th: We transfected 293t cells.</br>
-th: We collected cells and extracted RNA from then.</br>
-th: We did RT-PCR and qPCR.</br>
-</span>
-</h3>
-</div>
-</div>
-<div class="ss-row ss-medium">
-<div class="ss-left">
-<h3>
-<a>WEEK 6</a>
-<span>27th-31th, May</span>
-</h3>
-</div>
-<div class="ss-right">
-<h3>
-<a>siRNA screening (Failed)</a>
-<span>
-th: We cultured the 293t cells in 2 12-well plates.</br>
-th: We transfected 293t cells, with lipofectamine 2000.</br>
-th: We collected cells and extracted RNA from them.</br>
-th: We continued to extract RNA, and examine the concentration of total RNA. Then we did RT-PCR.</br>
-st : We did qPCR.</br>
-</span>
-</h3>
-</div>
-</div>
 	            <div class="ss-row">
                      <div class="ss-left">
-                     <h2 id="June">June</h2>
+                     <h4 id="June">Plasmids extraction</h4>
                      </div>
                      <div class="ss-right">
-                     <h2>2013</h2>
+                     <h4>Plasmids extraction with small quantity</h4>
                      </div>
                      </div>
@@ Line 798: / Line 764: @@
 <div class="ss-left">
 <h3>
-<a>WEEK 7</a>
+<a>Plasmids extraction</a>
-<span>19th-24th, June</span>
+<span>
+Preparation</br>
+.Column equilibration:  place a Spin Column CP5 into 50 ml collection tube (supplied in the kit) and add 2.5 ml Buffer BL to Spin Column CP5. Centrifuge for 2 min at  8,000 rpm (~8,228 × g).  Discard the flow-throw, and place Spin Column CP5 into the same collection tube.
+</br>2.Composition of solution:
+</br>   a)Solution I: 50 mM Glucose / 25 mM Tris-Cl / 10 mM EDTA，pH 8.0
+</br>   b)Solution II: 0.2 N NaOH / 1% SDS；
+</br>   c)Solution III: 3 M potassium acetate/ 2 M acetic acid
+</br></br>Protocol
+</br>1.Inoculate 1L LB/ampicillin (50μg/mL) medium placed in a 5 liter culture flask with E.coli carrying desired plasmid and grow at 37/C with agitation for 12-16 hr.
+</br>2.Pellet up to 1-2L bacteria in appropriate vessels by centrifugation at 4,000 rpm for 15 min at 4°C.
+</br>3.Decant or aspirate medium and discard. Add 18 mL Solution I. Resuspend cells completely by vortexing or pipetting up and down. Complete resuspension of cell pellet is vital for obtaining good yield.
+</br>4.Add 40 mL Solution II, gently mix by inverting and rotating tube several times. Incubate 10 minutes at room temperature.
+</br>5.Add 30 mL chilled Solution III, cover, and gently mix by inverting tube several times until a flocculent white precipitate forms. Incubate on ice for 10 minutes.
+</br>6.Centrifuge at 7000rpm for 15 minutes at 4oC to pellet the cellular debris and genomic DNA.
+</br>7.Transfer the supernatant into CS1 filter.
+</br>8.Measure the volume of the flow-through and add 0.6 volume of the isopropyl alcohol. Mix by inverting the bottle 15-25 times. Incubate on ice for 10 minutes.
+</br>9.Centrifuge at 7000rpm for 15min. Carefully decant the supernatant without disturbing the pellet..
+</br>10.Resuspend pelleted bacterial cells in 8 ml Buffer P1.
+Note: Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of the pellet.
+</br>11.Add 8 ml Buffer P2 and mix thoroughly by inverting the tube 6-8 times, and incubating at room temperature for 5 min.
+</br>    Note: Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min.
+</br>12.Add 8 ml Buffer P4 and mix immediately and thoroughly by inverting the tube 6-8 times, incubate at room temperature for 10 min. The solution should become cloudy.
+</br>　　Note: To avoid localized precipitation, mix the solution thoroughly, immediately after addition of Buffer P4.
+Centrifuge for 10 min at 8,000 rpm (~8,228 × g). A compact white pellet will form.
+</br>13.Note: If use more than 100 ml bacterial culture, prolong centrifugal time to 20-30 min.
+</br>14.Transfer the lysate into the Filtration Column CS. Gently insert the plunger into the Filtration
+Column CS and filter the cell lysate into a new 50 ml tube (not supplied in the kit).
+</br>15.Add 0.3 volume isopropanol to the cleared lysate, sealed the received tube, and mix completely and then transfer all solution to the Spin Column CP5. Centrifuge for 2 min at 8,000 rpm (~8,228 × g).
+</br>　　Note:  If the volume of isopropanol–lysate mixture is larger than the capacity of the column, it can be loaded the column two times.
+</br>16. Discard the flow-through and place the Spin Column CB5 back into the same collection tube.</br>
+. Add 10 ml Buffer PW to the column and centrifuge at 8,000 rpm (~8,228 × g) for 2 min. Discard the flow-through and place the Spin Columns CB5 back into the same collection tube.
+</br>18. Repeat step17.
+</br>19. Add 3ml 96%-100% ethanol to the Spin Column CP5 (put the CP5 in a collection  tube). Centrifuge for 2 min at  8,000 rpm (~8,228 × g).
+</br>20. Discard the flow-through and centrifuge at 8,000 rpm (~8,228 × g) for an additional 5 min for removing residual ethanol.
+</br>　　Note: Residual ethanol will influence the subsequent enzymatic reaction and sequence. Place the column with cap open in air for several minutes to dry the membrane.
+</br>21. To elute DNA, place the column in a clean 50 ml collection tube(supplied in the kit) and add 1-2 ml Buffer TB to the center of the membrane and incubate 5 min at room temperature, centrifuge at 8,000 rpm (~8,228 × g) for 2 min.
+</br>　　Note: Repeat step 14 to increase plasmid callback efficiency.If the volume of eluted buffer is less than 1 ml, it may affect recovery efficiency. The pH value of eluted buffer will have some influence in eluting. Buffer TB or distilled water (pH 7.0-8.5) is suggested to elute plasmid DNA. For long-term storage of DNA, eluting in Buffer TB and storing at  –20°C is recommended, since DNA stored in water is subject to acid hydrolysis.
+</span>
 </h3>
 </div>

Team:NJU China/Protocol

From 2013.igem.org

Revision as of 14:56, 27 September 2013

Protocol

Subculturing Cells

Plasmind DNA Transfection

RNA Extraction

RT-PCR

qPCR

Plasmids transformation

qPCR 1. According to the manufacturer’s instruction, use tubes to compound these reagent that listed below. All procedures were carried out on ice. 2. Set the PCR as requested below. Then, start to reverse-transcribe the target RNA

Plasmids extraction

Plasmids extraction with small quantity

siRNA screening (eliminated 308 siRNA, 467 siRNA and 516 siRNA seem to have good effect) 19th: We cultured 293t cells in 12-well plates. 20th: We transfected 293t cells. 21st: We collected cells and preserved in Trizol. 22nd: We extracted RNA from cells and did RT-PCR. 24th: We did qPCR.

July

2013

WEEK 8 19th-24th, June

July

2013

WEEK 8 19th-24th, June

WEEK 9 AND WEEK 10 16th -21th,22th-28th,July

August

2013

WEEK 11 29th July-4th, August

WEEK 12 5th-12th, August

WEEK 13 12th-18th, August

WEEK 14 19th-25th, August

September

2013

WEEK 15 26th August-1th September

Redoing the experiment on 23th August 31th: We extract RNA from brain and serum we collected on 22th August. 1th: We did RT-PCR, Q-PCR with the RNA we extracted on 31th August.

WEEK 16-17 2th-11th, September

qPCR 1. According to the manufacturer’s instruction, use tubes to compound these reagent that listed below. All procedures were carried out on ice.

2. Set the PCR as requested below. Then, start to reverse-transcribe the target RNA

siRNA screening (eliminated 308 siRNA, 467 siRNA and 516 siRNA seem to have good effect) 19th: We cultured 293t cells in 12-well plates.
20th: We transfected 293t cells.
21st: We collected cells and preserved in Trizol.
22nd: We extracted RNA from cells and did RT-PCR.
24th: We did qPCR.

Redoing the experiment on 23th August 31th: We extract RNA from brain and serum we collected on 22th August.
1th: We did RT-PCR, Q-PCR with the RNA we extracted on 31th August.