미생물 및 생물공학기초실험 5주차: Virus Titration by Plaque Assay [A+ 리포트]

소개

연세대학교 2017년 1학기 A+ 받은 리포트입니다.

실험관련 topic에 대해 많은 조사를 했기에 이 자료 하나로 충분할거에요.

목차

1. OBJECTIVE
2. MATERIALS
3. PROCEDURE
4. RESULTS
5. DISCUSSION/CONCLUSION
6. QUIZ
7. FURTHER STUDY
8. REFERENCE

본문내용

Objective
In this investigation, the main objective is to perform plaque assay by preparing serial
dilution of influenza virus in order to calculate the titration in plaque forming unit (pfu)/mL

Materials
 6-well plate, MDCK cells, serum-free MEM, phosphate buffered saline (PBS), virus
stock solution, Trypsin, 2X DMEM, 2% low-melting agar, pipettes and tips, e-tubes,
conical tubes and rack

Procedure
1. Prepare MDCK cells into each well of 6-well plate
2. Prepare 10-fold serial dilution of influenza virus (10-4, 10-5, 10-6) in PBS
3. From 6-well plate, remove the media carefully so that cells won’t fall apart
[careful not to scratch cells with pipette]
4. Wash with 1mL of PBS per well
5. Inoculate 250μL of virus per well and incubate for 30 minutes at room temp. (RT)
6. Remove the virus inoculums and wash with 1mL of PBS per well
7. Overlay 2mL of agarose mixture (2X DMEM 10mL + 2% agarose 10mL +
trypsin 80μL) per well and allow the agarose to solidify
8. Incubate at 37°C & 5% CO2 for 48~72hrs until plaques are formed

참고자료

· Experiment in Microbiology and Biochemical Engineering Laboratory Booklet – Department of Biotech. Yonsei University.
· Hamamoto I, Takaku H, Tashiro M, Yamamoto N (2013) High Yield Production of Influenza Virus in Madin Darby Canine Kidney (MDCK) Cells with Stable Knockdown of IRF7. PLoS ONE 8(3): e59892. doi:10.1371/journal.pone.0059892
· https://synapse.koreamed.org/DOIx.php?id=10.4167%2Fjbv.2013.43.2.148
· M.Matrosovich, T.Matrosovich, W.Garten, H.-D.Klenk. (2006). New low-viscosity overlay medium for viral plaque assays. Virology Journal 3:63.
· http://www.lonza.com/products-services/bio-research/cell-cultureproducts/specialty-media/per-c6-media.aspx
· http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/phage/plaques.html