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
