Protocol for treatment:
1. Added 10 ul DNase 10X Buffer to each 100 ul of RNA.
2. Added 2 ul Turbo DNase to each RNA.
3. Incubated at 37 C for 30 minutes
4. Added 10 ul DNase inhibitor to each sample
5. Incubated at room temp for 5 minutes, flicking occasionally
6. Centrifuged at 10,000 g for 1.5 minutes
7. Decanted the top 100 ul of supernatant to fresh tube
8. Placed in -80 until I could run qPCR
Later this afternoon I ran the qPCR on the treatment.
Reagent work sheet:
| Volume | Reactions X12 | |
| Ssofast Evagreen MM | 10 | 140 |
| FWD Actin Primer | 0.5 | 7 |
| REV Actin Primer | 0.5 | 7 |
| Nuclease Free H2O | 9.8 | 137.2 |
| RNA | 0.2 |
1. Added each from greatest volume to least to make the master mix.
2. Pipetted 21 ul master mix into each well of a qPCR partial plate
3. Added 0.2 ul sample to each tube
4. Positive control was DNA extract from Oly seed oysters
5. Negative control had nothing added to the master mix
I ran the following program:
| Sybr New Plate+Sybr cDNA 55 melt 2 | ||
| Step | Temperature | Time |
| Initiation | 95 C | 10 min |
| Elongation | 95 C | 15 sec |
| 55 C | 15 sec | |
| Read | ||
| 72 C | 15 sec | |
| Read | ||
| Repeat Elongation 40 times | ||
| Termination | 95 C | 1 min |
| 55 C | 1 sec | |
| Melt Curve Manual ramp 0.2C per sec Read 0.5 C | 65 - 95 C | 30 sec |
| 21 C | 10 min | |
| End |
You can see the results below
bright green = positive control, red = negative control, light blue down to brown = HM1-5, dark blue to yellow = SM1-5
The positive control began amplifying at the 21 cycle marks. One sample began at 30 cycles the rest were between 34 and 36.
You can see the raw datafile here.
Tomorrow I will isolate the RNA from the 310 heat shock samples, treat with DNase, and qPCR for quality.
A few notes:
ReplyDelete- Probably not a big deal, but you should've used 22uL of DNase Inactivation Reagent (protocol says to use 0.2 volumes if you used 2uL of DNase).
- You should specify which DNA sample you used for your positive control (e.g. label, date isolated, concentration)
- Your qPCR reaction doesn't add up. The Sso Fast is a 2x master mix. So, if you're using 10uL of Sso Fast, your final reaction volume (including template) should be 20uL. In this instance, it's not too big of deal, but for future reference this could impact your reactions if you deviate too far from the appropriate dilution.
- You mentioned a negative control, but you didn't actually run a negative control. To clarify, a negative control reaction would receive DNA template that you know will not produce amplification. Then, there's a no template control, in which you substitute water for template in the reaction. In your case, you actually don't have either because the reaction you label as a negative control isn't the same volume (because it's lacking template or water in place of template) as all the other reactions on your plate.
- You need to add your qPCR plate layout to your notebook.
- You need to add a link to the raw qPCR data file to your notebook, not a CSV export. The raw datafile is of file type TAD.