Daniel:Notebook/ComboLock/2017-2-14: Difference between revisions
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So what this tells me is that at the 350 ng range (taking direct from a 10 uM stock) is WAY too much. Even for the C2 amplification, which did have a CT, the CT was basically gibberish. This was probably caused by too much template getting in the way of primer binding. At approximately 1000X less (350 pg) this worked fine for all samples. A 100X dilution (3.5 ng) was fine for the C probes but failed for template. This is good to keep in mind when including positive controls in the future. This also explains several other times I have used positive controls and failed in previous experiments. Including [[Daniel:Notebook/ComboLock/2017-2-13|yesterday]]. | So what this tells me is that at the 350 ng range (taking direct from a 10 uM stock) is WAY too much. Even for the C2 amplification, which did have a CT, the CT was basically gibberish. This was probably caused by too much template getting in the way of primer binding. At approximately 1000X less (350 pg) this worked fine for all samples. A 100X dilution (3.5 ng) was fine for the C probes but failed for template. This is good to keep in mind when including positive controls in the future. This also explains several other times I have used positive controls and failed in previous experiments. Including [[Daniel:Notebook/ComboLock/2017-2-13|yesterday]]. | ||
So, looking at yesterday's results in the new lens, we can make a few more conclusions | So, looking at yesterday's results in the new lens, we can make a few more conclusions: | ||
*It now makes perfect sense why the template fraction always failed. From the calculation in [[Daniel:Notebook/ComboLock/2017-2-13|yesterday]]'s discussion, there is way more template than needed to bind to the 2 uL beads that I use. So there is still around 90% template in solution, which corresponds to 315 ng in my experiment, which will always fail. This also means the C probe binds efficiently, since the fraction was reduced to a measurable amount in the supernatant of the C probe fraction. See below. | *It now makes perfect sense why the template fraction always failed. From the calculation in [[Daniel:Notebook/ComboLock/2017-2-13|yesterday]]'s discussion, there is way more template than needed to bind to the 2 uL beads that I use. So there is still around 90% template in solution, which corresponds to 315 ng in my experiment, which will always fail. | ||
*This also means the C probe binds efficiently, since the fraction was reduced to a measurable amount in the supernatant of the C probe fraction. See below. | |||
==C Probe Binding Efficiency== | ==C Probe Binding Efficiency== |
Revision as of 20:34, 14 February 2017
Beads Binding Test (Started Thursday 2-9)
Positive Control Test
Yesterday's results showed that the positive controls failed time and time again. Looking at the math again, by adding 2 uL 10 uM template to the positive control reactions, I was adding >100X the amount Kapa's protocol recommends. So this time I ran a dilution series that covers each of the positive controls in dilutions of undiluted to 1:10000.
- qPCR
- Make qPCR master mixes according to following recipes Master Mix Recipes
- Add 48 uL of appropriate master mix and 2 uL sample according to plate layout
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x45
- 72C 2 min
- 16C hold
Master Mix | Primer Pair | Master Mix NoX | Forward Primer Amt (10 uM) | Reverse Primer Amt (10 uM) | uL nfH2O | uL 2X Kapa SYBR Fast | Total |
MMT | PCCT-F/PCCT-R | 11.2 | 11.2 | 11.2 | 235.2 | 280 | 537.6 |
MMC1 | primer24/primer4RC | 11.2 | 11.2 | 11.2 | 235.2 | 280 | 537.6 |
MMC2 | primer6/primer12RC | 11.2 | 11.2 | 11.2 | 235.2 | 280 | 537.6 |
Plate Layout
Note: Blank indicates lanes that were measured but had nothing (even master mix) in them.
Results
Averaged CT Values and Differential Table
Name | Well 1 | Well 2 | Average CT | Abs(Difference) | Cycles Above NTC |
Template-350 ng | 2 | 3 | NA | NA | NA |
Template-35 ng | 4 | 5 | NA | NA | NA |
Template-3.5 ng | 6 | 7 | NA | NA | NA |
Template-350 pg | 8 | 9 | 6.81 | 0.18 | 19.8 |
Template-35 pg | 12 | 13 | 10.66 | 0.07 | 16.0 |
C1-350 ng | 14 | 15 | NA | NA | NA |
C1-35 ng | 16 | 17 | NA | NA | NA |
C1-3.5 ng | 18 | 19 | 7.53 | 0.2 | 22.8 |
C1-350 pg | 23 | 24 | 12.04 | 1.24 | 18.3 |
C1-35 pg | 25 | 26 | 15.10 | 0.14 | 15.2 |
C2-350 ng | 27 | 28 | 37.81 | 0.32 | |
C2-35 ng | 29 | 30 | 5.75 | 0.37 | 25.2 |
C2-3.5 ng | 33 | 34 | 7.72 | 0.12 | 23.2 |
C2-350 pg | 35 | 36 | 11.02 | 0.02 | 19.9 |
C2-35 pg | 37 | 38 | 14.98 | 0.13 | 15.9 |
NTC-Template | 10 | 10 | 26.61 | 0 | |
NTC-C1 | 11 | 11 | 30.34 | 0 | |
NTC-C2 | 20 | 20 | 30.91 | 0 |
Graph
File:PosControlDilutions-20170214.png
Discussion
So what this tells me is that at the 350 ng range (taking direct from a 10 uM stock) is WAY too much. Even for the C2 amplification, which did have a CT, the CT was basically gibberish. This was probably caused by too much template getting in the way of primer binding. At approximately 1000X less (350 pg) this worked fine for all samples. A 100X dilution (3.5 ng) was fine for the C probes but failed for template. This is good to keep in mind when including positive controls in the future. This also explains several other times I have used positive controls and failed in previous experiments. Including yesterday.
So, looking at yesterday's results in the new lens, we can make a few more conclusions:
- It now makes perfect sense why the template fraction always failed. From the calculation in yesterday's discussion, there is way more template than needed to bind to the 2 uL beads that I use. So there is still around 90% template in solution, which corresponds to 315 ng in my experiment, which will always fail.
- This also means the C probe binds efficiently, since the fraction was reduced to a measurable amount in the supernatant of the C probe fraction. See below.