Cell and Supernatant qPCR of Myd88 and Dv-1 Cell Samples from 12-Well Plate Infection Experiment

Using samples from this extraction from the Feb. 8th infection experiment.

I will not be doing qPCR on every sample from this experiment. To cut down on what should be done, not every day 0 sample will be assayed. The table below shows which samples were used for the experiment, what the treatment conditions were, and what the Qubit concentrations of the DNA extracts are. I also added in the original inoculation fluid so that could be assayed (40ul sample). The spreadsheet with this information can be found here.

extraction_number plate_day infection cell_type sample_type qubit_concentration
1 0 yes Dv1 supernatant too low
2 0 yes Myd88 supernatant too low
3 0 no Dv1 supernatant too low
4 0 no Myd88 supernatant too low
13 0 yes Dv1 cells too low
14 0 yes Myd88 cells too low
15 0 no Dv1 cells too low
16 0 no Myd88 cells too low
25 5 yes Dv1 supernatant too low
26 5 yes Myd88 supernatant 3.4
27 5 no Dv1 supernatant 3.55
28 5 no Myd88 supernatant 2
29 5 yes Dv1 supernatant too low
30 5 yes Myd88 supernatant 3.22
31 5 no Dv1 supernatant 3.43
32 5 no Myd88 supernatant 5.78
33 5 yes Dv1 supernatant 6.7
34 5 yes Myd88 supernatant 6.67
40ul original sample NA NA innubila supernatant too low
36 5 no Myd88 supernatant 6.57
37 5 yes Dv1 cells 7.52
38 5 yes Myd88 cells 11
39 5 no Dv1 cells 6
40 5 no Myd88 cells 15.7
41 5 yes Dv1 cells 9.9
42 5 yes Myd88 cells 13.1
43 5 no Dv1 cells 6.83
44 5 no Myd88 cells 13.2
45 5 yes Dv1 cells 9.42
46 5 yes Myd88 cells 10.1
47 5 no Dv1 cells 9.47
48 5 no Myd88 cells 8

Dilution

  • For all the samples that had a readable DNA quant, I wanted to normalize them to 1ng/ul to input into the qPCR
  • For the too low samples, I acted like they were are 1ng/ul, but there is no way to tell
  • All samples were thawed on ice, vortexed, and spun down before dilution
extraction_number ul for 1ng/ul in 20 ul ul Hydration solution
1 NA NA
2 NA NA
3 NA NA
4 NA NA
13 NA NA
14 NA NA
15 NA NA
16 NA NA
25 NA NA
26 5.9 14.1
27 5.6 14.4
28 10.0 10.0
29 NA NA
30 6.2 13.8
31 5.8 14.2
32 3.5 16.5
33 3.0 17.0
34 3.0 17.0
40ul original sample NA NA
36 3.0 17.0
37 2.7 17.3
38 1.8 18.2
39 3.3 16.7
40 1.3 18.7
41 2.0 18.0
42 1.5 18.5
43 2.9 17.1
44 1.5 18.5
45 2.1 17.9
46 2.0 18.0
47 2.1 17.9
48 2.5 17.5

Supernatant qPCR

  • The first plate would just be the supernatant samples
  • Rows A, B, C, and D will get the 115 primer (DiNV)
  • Rows E, F, G, and H will get the lambda primer (extraction control)
  • All DNA, primers, and Supermix (Sso) were thawed on ice, vortexed, and spun down before use
  • A master mix for each primer was made on ice
  • Each primer has 48 samples, plus 4 for error, ends with 52 = n
  • 115 master mix:
    • 5ul Sso * 52 = 260ul
    • 0.5ul 115 F * 52 = 26ul
    • 0.5ul 115 R * 52 = 26ul
    • 3ul nuclease free water * 52 = 156ul
  • The master mix was vortexed, spun down, and kept on ice
  • Lambda master mix:
    • 5ul Sso * 52 = 260ul
    • 0.5ul Lambda F * 52 = 26ul
    • 0.5ul Lambda R * 52 = 26ul
    • 3ul nuclease free water * 52 = 156ul
  • The master mix was vortexed, spun down, and kept on ice
  • A qPCR plate and seal were used
  • 9ul of each master mix was added to each of the planned wells (see plate layout)
  • 1ul of DNA was added to each reaction well, with each sample getting triplicate wells per primer
  • Each well was pipette mixed with a multichannel pipette set to 5ul
  • The plate was sealed, and spun down for 3 minutes at 3000rcf
  • The plate was put in the qPCR machine:
    • Used program CFX manager
    • Selected user-defined protocol, exisiting protocol, and KMM folder
    • Selected the p47 program
    • Used the machine buttons to open and close the lid
    • Started the program
  1 2 3 4 5 6 7 8 9 10 11 12  
A 1 1 1 2 2 2 3 3 3 4 4 4  
B 25 25 25 26 26 26 27 27 27 28 28 28 115
C 29 29 29 30 30 30 31 31 31 32 32 32  
D 33 33 33 34 34 34 40ul sample 40ul sample 40ul sample 36 36 36  
E 1 1 1 2 2 2 3 3 3 4 4 4  
F 25 25 25 26 26 26 27 27 27 28 28 28 lambda
G 29 29 29 30 30 30 31 31 31 32 32 32  
H 33 33 33 34 34 34 40ul sample 40ul sample 40ul sample 36 36 36  

Cells qPCR

  • The first plate is only the cell samples
  • Rows A, B, C, and D will get the 115 primer (DiNV)
  • Rows E, F, G, and H 1-6 will get the RPL11 primer (Dv-1) and rows E, F, G, and H 7-12 will get the RP49 primer (melanogaster)
  • All DNA, primers, and Supermix (Sso) were thawed on ice, vortexed, and spun down before use
  • A master mix for each primer was made on ice
  • The 115 primer has 48 samples, plus 4 for error, ends with 52 = n
  • 115 master mix:
    • 5ul Sso * 52 = 260ul
    • 0.5ul 115 F * 52 = 26ul
    • 0.5ul 115 R * 52 = 26ul
    • 3ul nuclease free water * 52 = 156ul
  • The master mix was vortexed, spun down, and kept on ice
  • The two cell control primers has 24 samples, plus 2 for error, ends with 26 = n
  • RPL11 master mix:
    • 5ul Sso * 26 = 130ul
    • 0.5ul RPL11 F * 26 = 13ul
    • 0.5ul RPL11 R * 26 = 13ul
    • 3ul nuclease free water * 26 = 78ul
  • The master mix was vortexed, spun down, and kept on ice
  • RP49 master mix:
    • 5ul Sso * 26 = 130ul
    • 0.5ul RP49 F * 26 = 13ul
    • 0.5ul RP49 R * 26 = 13ul
    • 3ul nuclease free water * 26 = 78ul
  • The master mix was vortexed, spun down, and kept on ice
  • A qPCR plate and seal were used
  • 9ul of each master mix was added to each of the planned wells (see plate layout)
  • 1ul of DNA was added to each reaction well, with each sample getting triplicate wells per primer
  • Each well was pipette mixed with a multichannel pipette set to 5ul
  • The plate was sealed, and spun down for 3 minutes at 3000rcf
  • The plate was put in the qPCR machine:
    • Used program CFX manager
    • Selected user-defined protocol, exisiting protocol, and KMM folder
    • Selected the p47 program
    • Used the machine buttons to open and close the lid
    • Started the program
  1 2 3 4 5 6 7 8 9 10 11 12  
A 13 13 13 14 14 14 15 15 15 16 16 16  
B 37 37 37 38 38 38 39 39 39 40 40 40 115
C 41 41 41 42 42 42 43 43 43 44 44 44  
D 45 45 45 46 46 46 47 47 47 48 48 48  
E 13 13 13 15 15 15 14 14 14 16 16 16  
F 37 37 37 39 39 39 38 38 38 40 40 40  
G 41 41 41 43 43 43 42 42 42 44 44 44  
H 45 45 45 47 47 47 46 46 46 48 48 48  
      RPL11           RP49        

Data from plate 1 (supernatant) can be found here
Data from plate 2 (cells) can be found here
Analysis of these samples can be found here