qPCR
qPCR
Write a short report of the qPCR experiment including how you set it up, what fluorophore was used and how it works. Describe the results of the analysis including the concentration of your starting sample and the # cycles where it passes threshold.
To make the standard curve was made by serial dilutions.
Human control DNA with known concentration was used:
1. First was diluted 1 to 11 and the concentration was 1ug in 5ul
2. 1ul of previous dilution in 9ul H2O > 0.01ug DNA in 5ul
3. 1ul of previous dilution in 9ul H2O > 0.001ug DNA in 5ul
4. 1ul of previous dilution in 9ul H2O > 0.0001ug DNA in 5ul
5. 1ul of previous dilution in 9ul H2O > 0.00001ug DNA in 5ul
To make my DNA sample I used dilutions:
1. 1 to 10
2. 1 to 100
The master mix :
Nuclease free H2O 19ul
2x SYBR Mastermix with polymerse 25ul
L primer (100pM/ul) 0.5ul
R primer (100pM/ul) 0.5ul
DNA template 5ul
Total volume 50ul
For the blank instead of DNA template was used nuclease free H2O
The standard and samples are set up in the plate in order and assigned in the computer program.
The protocol of PCR is HV1. Cycling conditions are: 40 cycles of 30 seconds at 95oC, 30 seconds at 53oC and 30 seconds at 72oC. There is an initial 3 minute heating at 95oC to activate the polymerase and an additional 7 minute extension step at the end. Samples are kept at 4oC until taken out of the PCR machine.
SYBR green was used and it binds to double-stranded DNA by intercalating between base pairs, and fluoresces only when bound to DNA. The detection occurs during the PCR cycle at the end of either the annealing or the extension step when the greatest amount of double-stranded DNA product is present.
The standard curve was good beside the last two standards (highest) were of the line. All the samples were on the standard.
The computer provides us with diagram for standard curve and amplification curve and where the sample pass the detection threshold. Also the program provides
Write a short report of the qPCR experiment including how you set it up, what fluorophore was used and how it works. Describe the results of the analysis including the concentration of your starting sample and the # cycles where it passes threshold.
To make the standard curve was made by serial dilutions.
Human control DNA with known concentration was used:
1. First was diluted 1 to 11 and the concentration was 1ug in 5ul
2. 1ul of previous dilution in 9ul H2O > 0.01ug DNA in 5ul
3. 1ul of previous dilution in 9ul H2O > 0.001ug DNA in 5ul
4. 1ul of previous dilution in 9ul H2O > 0.0001ug DNA in 5ul
5. 1ul of previous dilution in 9ul H2O > 0.00001ug DNA in 5ul
To make my DNA sample I used dilutions:
1. 1 to 10
2. 1 to 100
The master mix :
Nuclease free H2O 19ul
2x SYBR Mastermix with polymerse 25ul
L primer (100pM/ul) 0.5ul
R primer (100pM/ul) 0.5ul
DNA template 5ul
Total volume 50ul
For the blank instead of DNA template was used nuclease free H2O
The standard and samples are set up in the plate in order and assigned in the computer program.
The protocol of PCR is HV1. Cycling conditions are: 40 cycles of 30 seconds at 95oC, 30 seconds at 53oC and 30 seconds at 72oC. There is an initial 3 minute heating at 95oC to activate the polymerase and an additional 7 minute extension step at the end. Samples are kept at 4oC until taken out of the PCR machine.
SYBR green was used and it binds to double-stranded DNA by intercalating between base pairs, and fluoresces only when bound to DNA. The detection occurs during the PCR cycle at the end of either the annealing or the extension step when the greatest amount of double-stranded DNA product is present.
The standard curve was good beside the last two standards (highest) were of the line. All the samples were on the standard.
The computer provides us with diagram for standard curve and amplification curve and where the sample pass the detection threshold. Also the program provides