Doing qPCR to Compare DiNV Levels in Hirt Extracted DNA and Puregene Extracted DNA
Using DNA samples from Hirt Extraction and Puregene Extraction. These DNA are from the exact same samples, just with different extraction methods. The goal is to see if the Hirt method does give a better ratio of DiNV DNA to Dv-1 cell DNA. Samples were normalized to 1ng/ul before use in qPCR. DiNV primers are called 115, and Dv-1 cell primers are RPL11.
DNA dilution to 1ng/ul
- All samples were kept on ice and/or thawed on ice
| Tube number | Sample | Sample DNA concentration | ul DNA | ul Molec grade water for 1ng/ul concentration |
|---|---|---|---|---|
| 1 | Hirt 1.5-2 | 63.7ng/ul | 2ul | 127.4ul |
| 2 | Hirt 0.75 | 35.5ng/ul | 2ul | 71ul |
| 3 | Hirt 0.5-C | 130ng/ul | 2ul | 260ul |
| 4 | Puregene 1.5-2 | 3.72ng/ul | 3ul | 11.16ul |
| 5 | Puregene 0.75 | 3.57ng/ul | 3ul | 10.71ul |
| 6 | Puregene 0.5-C | 26.1ng/ul | 2ul | 52.2ul |
qPCR setup
- I have 6 samples, which need to be run in triplicate for 2 primers, so that will be 6 * 3 * 2 = 36 separate reactions
- However I will need to make 2 separate mastermixes for the two primers
- Kent suggested starting the machine and the computer before starting the process to make sure no one takes the computer before you need it
- Turn on qPCR machine so it can warm up
- Log into computer
- Click on the Biorad CFX manager
- Click user defined, then select existing, then KMM, then p47 program
- Using SsoAdvanced Universal SYBR Green Supermix as the reagent for the qPCR
- Make master mixes on ice
- RPL11 master mix:
- 5ul Sso * 21 = 105ul
- 0.5ul RPL11 F * 21 = 10.5ul
- 0.5ul RPL11 R * 21 = 10.5ul
- 3ul molec grade water * 21 = 63ul
- 115 master mix
- 5ul Sso * 21 = 105ul
- 0.5ul 115 F * 21 = 10.5ul
- 0.5ul 115 R * 21 = 10.5ul
- 3ul molec grade water * 21 = 63ul
- Vortexed and spin down mixes
- Added 9ul of RPL11 mix to 18 wells A1-9 and B1-9
- Added 9ul of 115 mix to 18 wells C1-9 and D1-9
- Added 1ul DNA to those wells (see table), where the number corresponds to the tube number in the above table
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| A | 1 | 1 | 1 | 2 | 2 | 2 | 3 | 3 | 3 | |||
| B | 4 | 4 | 4 | 5 | 5 | 5 | 6 | 6 | 6 | |||
| C | 1 | 1 | 1 | 2 | 2 | 2 | 3 | 3 | 3 | |||
| D | 4 | 4 | 4 | 5 | 5 | 5 | 6 | 6 | 6 | |||
| E | ||||||||||||
| F | ||||||||||||
| G | ||||||||||||
| H |
- Put the plate seal on the plate and made sure to take off the perforated edges
- Spun down the plate in 4055 (I should have vortexed it before this)
- Pressed the open lid button on the machine
- Put the plate in
- Pressed the lid button again
- Pressed start on the computer
- Program runs for about an hour
- Saved the results to a new folder on the computer with my name
- After the run was done, exported all the files onto a flash drive to use on my computer
Analysis