qPCR of 115 and Lambda on D. innubila Concentrated and Non-concentrated Supernatant Fluid For Viral Quantification
Using DNA extracted from fluid concentration experiment to do 115 and Lambda qPCR to see if the concentration does increase the ammount of virus in these samples and by how much. The DNA extracts were Qubited but every sample was too low to read on the Broad Range kit meaning that it was below 2ng/ul concentration. Therefore I was not able to standardize the input amount of DNA, but the lambda value should be an internal standard for each reaction anyways. There are 8 samples that were run in triplicate for each primer, and the plate layout is below with sample number inside the plate layout:
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| A | 1 | 1 | 1 | 1 | 1 | 1 | ||||||
| B | 4 | 4 | 4 | 4 | 4 | 4 | ||||||
| C | 6 | 6 | 6 | 6 | 6 | 6 | ||||||
| D | 8 | 8 | 8 | 8 | 8 | 8 | ||||||
| E | 11 | 11 | 11 | 11 | 11 | 11 | ||||||
| F | 13 | 13 | 13 | 13 | 13 | 13 | ||||||
| G | 17 | 17 | 17 | 17 | 17 | 17 | ||||||
| H | 18 | 18 | 18 | 18 | 18 | 18 | ||||||
| 115 | lambda |
- Samples and reagents were thawed on ice, vortexed, and spun down before use
- There are 24 reactions per primer, with error, n = 26
- 115 master mix:
- 5ul Sso supermix * 26 = 130ul
- 0.5ul 115 F primer * 26 = 13ul
- 0.5ul 115 R primer * 26 = 13ul
- 3ul nuclease free water * 26 = 108ul
- The master mix was made on ice, vortexed, and spun down
- Lambda master mix:
- 5ul Sso supermix * 26 = 130ul
- 0.5ul Lambda F primer * 26 = 13ul
- 0.5ul Lambda R primer * 26 = 13ul
- 3ul nuclease free water * 26 = 108ul
- The master mix was made on ice, vortexed, and spun down
- I used the qPCR specific plates and seals
- 9ul of the appropriate master mix was added to the planned well (115 to columns 1, 2, and 3, and Lambda to columns 5, 6, and 7)
- 1ul of DNA was added to the planned well (see table above)
- Each well was pipette mixed with 5ul using a multichannel
- The plate was sealed and centrifuged for 2 minutes at 4,000g
- 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
- Data from the qPCR machine can be found here
- And analysis of the Cq results can be found here