Team:NJU China/Protocol

From 2013.igem.org

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<a>Experiment training</a>
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<a>Plasmind DNA Transfection</a>
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We learnt to culture the 293t cells.</br>
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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).
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We learnt to extract plasmids and transfect it into 293t cells.</br>
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</br>1. Adherent cells: One day before transfection, plate 0.5-2*105 cells in 500μl of growth medium without antibiotics so that cells will be 90%-95% confluent at the time of transfection.
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We learnt to collect exosomes.</br>
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Suspension cells: Just prior to preparing complexes, plate 4-8*105 cells in 500μl of growth medium without antibiotics.
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We learnt to extract RNA from exosomes and cells.</br>
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</br>2. For each transfection sample, prepare complexes as follows:
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We learnt to do RT-PCR and qPCR.
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</br>a. Dilute DNA in 50μl of Opti-MEM
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</br>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.
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</br>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.
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</br>3. Add the 100μl of complexes to each well containing cells and medium. Mix gently by rocking the plate back and forth.
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</br>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.
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</br>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.
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Scaling Up or Down Transfections
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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.
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</br><img src="https://static.igem.org/mediawiki/2013/2/2f/20130927_222700.jpg">
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</br>1Surface areas may vary depending on the manufacturer.
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</br>2Volumes of dilution medium in Step 2a&2b of DNA or RNAi transfection protocols.
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</br></br>Optimizing Plasmid DNA Transfection
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</br>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.
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                     <h2 id="April">April</h2>
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                     <h2 id="April">2</h2>
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Revision as of 14:29, 27 September 2013

 

Protocol

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 0.5-2*105 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 4-8*105 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.

2

2013

WEEK 2 14th-18th, April

siRNA screening (Failed) 14th: We extracted plasmids and cultured the 293t cells.
15th: We transfected plasmids into 293t cells.
16th: We collected cells and preserved it in Trizol.
17th: We extracted RNA, then did RT-PCR with the RNA.
18th: We did qPCR with the cDNA we got on 17th April.
21th: We reexamined the concentration of RNA, and redid qPCR.

WEEK 3 27th, April – 1st, May

siRNA screening (Failed) 27th: We cultured the 293t cells in a 12-well plate.
28th: We transfected 293t cells.
29th: We collected cells and extracted RNA from it.
30th: We did RT-qPCR with the RNA we extracted on 29th April.
1st: We did qPCR with the cDNA we got on 30th April.

May

2013

WEEK 4 8th-13th, May

siRNA screening (Failed), examination whether siRNA are capsulated into exosomes (Success) 8th: We extracted plasmids of 3 kinds of siRNA.
9th: We cultured 293t cells in eight D10 dishes and a 12-well plate.
10th: We extracted 2 kinds of over-expression plasmids and examined the concentration of it. Then we transfect these plasmids into 293t cells respectively.
11th: We collected cells and exosomes from 8 D10 dish, and cells from 12-well plate.
12th: We extracted RNA from cells and exosomes.
13th: We did RT-PCR and qPCR.

WEEK 5 15th-18th, May

siRNA screening (Failed) 15th: We cultured the 293t cells in a 12-well plate.
16th: We transfected 293t cells.
17th: We collected cells and extracted RNA from then.
18th: We did RT-PCR and qPCR.

WEEK 6 27th-31th, May

siRNA screening (Failed) 27th: We cultured the 293t cells in 2 12-well plates.
28th: We transfected 293t cells, with lipofectamine 2000.
29th: We collected cells and extracted RNA from them.
30th: We continued to extract RNA, and examine the concentration of total RNA. Then we did RT-PCR.
31st : We did qPCR.

June

2013

WEEK 7 19th-24th, June

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(lipo*2,467*2,516*2).
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(lipo*3,RVG*3,non-related plasmids*3).
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 plasmid*2, 516 plasmid*2).
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 (empty*3, 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.

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