I need to know what calculations I'm supposed to do in order to enter the appropriate values. All information/materials given are pasted below.
Following the amplification protocol, fill in the appropriate quantitation values, DNA input and TE input for the samples, reagent blanks and controls within theAmplification Worksheet.
Amplification Protocol:
Scope This protocol is used for the PHA 6851 amplification worksheet set-up. Procedure 1. Amplification Worksheet Set-up: a. Complete the PHA 6851 Amplification Worksheet with the reagent lot numbers. b. Tabulate the total number of reactions for the amplification including the DNA samples, a positive control, and a negative control and enter on the "# of samples" section of the PHA 6851 Amplification Worksheet. c. Calculate and record the total volume of each component needed by multiplying the volume of each component by the total number of reactions. i. PCR Reaction Mix - 7.5 ul per reaction ii. Primer Set - 2.5 ul per reaction d. Ensure the Quantitativesults, target values and the DNA and TE-4 buffer (diluent) volumes are entered on the amplification worksheet. i. The maximum DNA volume is 15ul. 2. Amplification parameters: a. Utilize the small autosomal quantitativelue for amplification. a. For samples with a degradation index of >4, utilize the large autosomal quantitati value for amplification. b. The target value for unknown samples is 0.75 ng. c. The target value for known samples is 0.5 ng d. For samples that result in less than 0.5ul to be pipetted, the sample must be diluted and the quantitativelue adjusted accordingly. e. A positive and negative amplification control shall be included for each amplification set-up of unknown and known samples. a. The positive control shall have 7.5ul of Control and 7.5ul of TE added b. The negative control shall have 15ul of TE added.Quantity M:F Degradation Sample Name Target Name CT ng/ul Ratio Index Knife handle IPC 26.89 Knife handle Large Autosomal 30.29 0.02 14.50 Knife handle Small Autosomal 28.54 0.29 14.50 Knife handle Y 28.18 0.23 Knife blade IPC 33.70 Knife blade Large Autosomal Jundetermined Knife blade Small Autosomal 26.02 1.55 Knife blade Y undetermined Cigarette Butt IPC 27.67 Cigarette Butt Large Autosomal 26.06 0.44 1.02 Cigarette Butt Small Autosomal 27.77 0.45 1.02 Cigarette Butt Y 27.28 10.44 Bathroom sink stain IPC 27.57 Bathroom sink stain Large Autosomal 23.89 1.89 0.72 Bathroom sink stain Small Autosomal 26.21 1.36 0.72 Bathroom sink stain Y 25.80 1.24 Q Reagent Blank IPC 27.73 Q Reagent Blank Large Autosomal undetermined Q Reagent Blank Small Autosomal undetermined Q Reagent Blank Y undeterminedQ Reagent Blank IPC 27.73 Q Reagent Blank Large Autosomal undetermined Q Reagent Blank Small Autosomal undetermined Q Reagent Blank Y undetermined Suspect Standard IPC 28.00 Suspect Standard Large Autosomal 24.34 1.39 1.06 Suspect Standard Small Autosomal 26.08 1.48 1.06 Suspect Standard Y 25.72 1.31 Victim Standard IPC 28.05 Victim Standard Large Autosomal 25.82 0.51 0.76 Victim Standard Small Autosomal 27.98 0.39 0.76 Victim Standard Y undetermined K Reagent Blank IPC 28.02 K Reagent Blank Large Autosomal undetermined K Reagent Blank Small Autosomal undetermined K Reagent Blank Y undeterminedReagents: Lot # Number of samples 12 Amplification Kit 185678 Reaction Mix Volume 90 ul TE4 181324 Primer Volume 30 ul Sample# Quant results Target ng DNA (PL) TE (UL) (ng/ul) 1. Knife handle 0.02 0.75 2. Knife blade 1.55 0.75 3. Cigarette Butt 0.45 0.75 4. Bathroom sink stain 1.36 0.75 5. Q Extraction Blank 6. Positive Q control 7.5 7.5 7. Negative Q control 15 8. Victim standard 0.39 0.5 9. Suspect Standard 1.48 0.5 10. K Extraction Blank 11. Positive K Control 7.5 7.5 12. Negative K Control 15
