Experiment training
We learnt to culture the 293t cells.
We learnt to extract plasmids and transfect it into 293t cells.
We learnt to collect exosomes.
We learnt to extract RNA from exosomes and cells.
We learnt to do RT-PCR and qPCR.
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.
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.
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.
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.
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.
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.
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.
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