Determination of TCID50 (50% tissue culture infectious dose) end point using Herpes simplex virus by Sperman-Karber formula

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2.0 Materials and method

2.1 Materials and instrumentation:

Write all materials and instrument used in all methods

For instance:

minimum essential medium eagle from Sigma, UK (M7278) (MEM) 500 ml,

foetal calf serum

glutamin essential amino acid

penicillin and streptomicyn

non essential amino acid

trypsin

Dulbecco’s phosphate buffered saline 100ml from Sigma, UK (D8537)

Trypan blue

And the other equipment like ( electronic pipit, haemocytometer, beaker, universal tube, pejo bottle, inverted microscope ……..etc

Also the materials for PCR

Vero cells: definition

Virus strain: HSV2 –st

Acyclovir

2.2 methods:

 

  • Cell culture

1.a growth medium preparation  (10% calf serum)

2.bsubculture of cells

2.ccell counting

2.dpreparation of cell cultures for inoculation (Vero cells)

 

  • Virus titration in cell culture

Determination of TCID50 (50% tissue culture infectious dose) end point using Herpes simplex virus by Sperman-Karber formula

2.apreparation of maintenance medium 500ml, 2% foetal calf serum

 

  • Taq man Real Time PCR

3.astandard curve preparation:

Create standard sequence for HSV1, HSV2, POLYOMA 9, BK VIRUS, CMV in one sequence then send it to Eurofine Genomic to insert it in the plasmid (pEX-K4-JA1) and synthesis the sequence by vector (e.gEcoli)

Calculate how many copies of viruses in stock concentration

http://cels.uri.edu/gsc/cndna.html( write the formula with referencing)

number of copies in stock = 4.78* 1011

then made serial dilution 1:10

the number of DNA copies in first dilution 10-1= 4.78*1011/1559/10=

then reduce one power each time

plot a curve on Excel by use the right equation and insert the CT value on X axis and the concentration( which equal the number of DNA copies) on Y axis to create standard curve for 10 standard concentrations , so we can use it to identify the unknown sample

run the PCR for 10-1 to 10-10

3.btaq-man PCR for 100 TCID50

 

  • Repeat the TCID50 use 100 tcid50 per well ( set 2 plates)

Add acyclovir (use the EC50 the concentration of ACV that inhibit 50% ) and lower and higher concentration

  • Taq-man PCR for well A from each concentration
  • Taq-man PCR for the first column given negative and the column before

 

Attachments:

Cell line data sheet for vero cells information

Cell culture procedure

TCID50 procedure

Plasmid information that used in standard curve + viruses sequences + standard sequence

Result table to use the CT figures

Taq-man Pcr procedure

Acyclovir information sheet

Instructions:

I want a native speaker to write this introduction, I’m not looking to find any grammar mistakes. I want to write all the materials and instrument for all methods in the beginning then write each method in paragraphs as dissertation’s style ,use of academic language, third person voice and use the transition words. The references must be in Harvard style. The work must be paraphrased, I don’t want you to copy and paste the information from any source because this will be considered as plagiarism.If there’s any further information needed please contact me

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