Doing qPCR on DNA Extracts of Dv-1 and Myd88 Cells, Infected with DiNV and Control

Because of the variability between the extraction replicates, and the number of PCRs I need to do, I am not using the extraction replicates for this process. Just one DNA sample per sample.

Sample inforation and plate planning is here and here

Diluting Samples

  • Want to have all samples at 1ng/ul to use for input
  • For the samples that are too low, I did not do anything with them and used them as-is for the qPCR
  • All samples were thawed on ice, vortexed and spun down before use
  • I made new dilution tubes for the samples needing a dilution, and used the DNA hydration solution from the Puregene DNA extraction kit to do the elution
Sample_ID qubit_quantity ul_EB_to_1ng/ul ul_DNA
1 too low NA NA
2 too low NA NA
3 too low NA NA
4 5.16 15.48 3
5 too low NA NA
6 48.1 96.2 2
7 13.2 26.4 2
8 44.3 88.6 2
9 10.6 21.2 2
10 45.6 91.2 2
11 11.2 22.4 2
12 20.1 40.2 2
13 14.5 29 2
14 18.3 36.6 2
15 14.2 28.4 2
16 14 28 2
  • All samples were kept on ice

qPCR Plates

  • For all samples:
    • 3 replicate reactions per PCR primer
    • Need to have 115 (DiNV) reactions
    • Need to have a cell-line specific reaction (either RP49 for Myd88 or RPL11 for Dv-1)
    • For supernatant samples only, need to have Lambda reaction
  • Because of the number of samples, this was spit up over 2 plates and were laid out like this:

Master Mixes Plate 1

  • I made 10uM aliquots of the Lambda (from Kent’s box) and RP49 ( number 8 and 9 on lab spreadsheet) primers
    • 10ul of 100uM stock
    • 90ul nuclease free water
  • Made master mixes for each plate separately (done a few hours apart)
  • All components were vortexed and spun down before use
  • Sample numbers for plate 1:
    • Lambda is 18 + 2 for error: 20
    • RPL11 is 18 + 2 for error: 20
    • 115 is 39 + 4 for error: 43
    • RP49 is 21 + 2 for error: 23
  • 115 master mix:
    • 5ul Sso (supermix) * 43 = 215ul
    • 0.5ul 115 F primer * 43 = 21.5ul
    • 0.5ul 115 R primer * 43 = 21.5ul
    • 3ul nuclease free water * 43 = 129ul
  • RP49 master mix:
    • 5ul Sso (supermix) * 23 = 115ul
    • 0.5ul RP49 F primer * 23 = 11.5ul
    • 0.5ul Rp49 R primer * 23 = 11.5ul
    • 3ul nuclease free water * 23 = 69ul
  • RPL11 master mix:
    • 5ul Sso (supermix) * 20 = 100ul
    • 0.5ul RPL11 F primer * 20 = 10ul
    • 0.5ul RPL11 R primer * 20 = 10ul
    • 3ul nuclease free water * 20 = 60ul
  • Lambda master mix:
    • 5ul Sso (supermix) * 43 = 100ul
    • 0.5ul Lambda F primer * 43 = 10ul
    • 0.5ul Lmbda R primer * 43 = 10ul
    • 3ul nuclease free water * 43 = 60ul
  • Vortexed and spun down mixes and kept on ice

Plate set up

  • Used specific qPCR plate and seal
  • Put 9ul of primer mix into correct wells (see plate picture above)
  • Added 1ul of sample to each well (see plate picture above)
  • Pipette mixed each well with 5ul using a multichannel
  • Spun down the plate for 1 min at 4000g

At the qPCR machine

  • Logged into computer
  • Clicked on the BioRAD CFX manager software
  • Chose user-defined protocol, existing protocol, and KMM folders
  • Selected the p47 program
  • Used the button on the lid to open the machine
  • Placed the plate in correct orientation
  • Pressed the button on the machine to close it
  • Started the program on the computer
  • Ran for 1.5ish hours, and saved the results on my folder on the computer and put on a flash drive

Master Mixes Plate 2

  • All components were vortexed and spun down before use again and kept on ice the whole time
  • Sample numbers for plate 2:
    • Lambda is 6 + 1 for error: 7
    • RPL11 is 6 + 1 for error: 7
    • 115 is 9 + 1 for error: 10
    • RP49 is 3 + 1 for error: 4
  • 115 master mix:
    • 5ul Sso (supermix) * 10 = 50ul
    • 0.5ul 115 F primer * 10 = 5ul
    • 0.5ul 115 R primer * 10 = 5ul
    • 3ul nuclease free water * 10 = 30ul
  • RP49 master mix:
    • 5ul Sso (supermix) * 4 = 20ul
    • 0.5ul RP49 F primer * 4 = 2ul
    • 0.5ul Rp49 R primer * 4 = 2ul
    • 3ul nuclease free water * 4 = 12ul
  • RPL11 master mix:
    • 5ul Sso (supermix) * 7 = 35ul
    • 0.5ul RPL11 F primer * 7 = 3.5ul
    • 0.5ul RPL11 R primer * 7 = 3.5ul
    • 3ul nuclease free water * 7 = 21ul
  • Lambda master mix:
    • 5ul Sso (supermix) * 7 = 35ul
    • 0.5ul Lambda F primer * 7 = 3.5ul
    • 0.5ul Lmbda R primer * 7 = 3.5ul
    • 3ul nuclease free water * 7 = 21ul
  • Vortexed and spun down mixes and kept on ice

Plate set up

  • Used specific qPCR plate and seal
  • Put 9ul of primer mix into correct wells (see plate picture above)
  • Added 1ul of sample to each well (see plate picture above)
  • Pipette mixed each well with 5ul using a multichannel
  • Spun down the plate for 1 min at 4000g

At the qPCR machine

  • Logged into computer
  • Clicked on the BioRAD CFX manager software
  • Chose user-defined protocol, existing protocol, and KMM folders
  • Selected the p47 program
  • Used the button on the lid to open the machine
  • Placed the plate in correct orientation
  • Pressed the button on the machine to close it
  • Started the program on the computer
  • Ran for 1.5ish hours, and saved the results on my folder on the computer and put on a flash drive