Daniel:Notebook/ComboLock/2016-8-22
Positive Control Amplicon Test (Started Thursday)[edit]
Protocol[edit]
- Ethanol Precipitation
- Centrifuge at 12000rpm at 4C for 25 minutes
- Remove supernatant and add 750 uL 70% EtOH
- Centrifuge at 12000rpm at 4C for 15 minutes
- Remove supernatant and let dry in PCR hood for 15 min
- Resuspend in 30 uL nfH2O
Nanodrop Results[edit]
Discussion[edit]
Looking back at the experiment, I am particularly going to highlight the results from Friday, specifically the gel results. I'm going to show it below as a reminder as well as the sample matrix from Thursday as a reference for the contents of the lanes.
Sample No | Sample Name | LatchX2 (300 ng - 2 uL 10 uM) | LatchX3 (300 ng - 2 uL 10 uM) | PCAmp1 | PCAmp3 | Expected Product |
1 | Amp1 Test | X | X | Amp1 | ||
2 | Amp3 Test | X | X | Amp3 | ||
3 | LatchX2 Specificity | X | X | X | Amp1 | |
4 | LatchX3 Specificity | X | X | X | Amp3 | |
5 | No Template | X | X | None | ||
6 | No Latch | X | X | None | ||
7 | No Padlock | X | X | X | X | None |
8 | PCAmp2 Only | X | X | None |
File:2016-08-19-PosControl-qPCR.png
Several things stand out to me. First is that 3 of the lanes show the expected product (231 bp). This is great news! However, the two lanes that show it strongly are lanes 3 and 4, which are the lanes that include two different beads and therefore the product may be ambiguous (we'll see when the sequencing results come back). The first lane shows some signal and the second lane shows none. These are the two lanes that should just work, they are the right template and latch combination. Since I technically added twice as much template in samples 3 and 4 this may have contributed to the increased product rate. It shouldn't since there was the same amount of correct template in samples 3 and 4 as in 1 and 2, but it is my best hypothesis at the moment.
The second thing to note is that all of the negative control lanes showed product except lane 7 (no padlock). So this leads me to believe that the primary cause of background signal is the amplification of unligated padlock or padlock ligated without latch present. If it is the first, that would mean that my exonuclease digestion is ineffective. If it is the latter, that means that an increased amount of latch should reduce the amount of background product since far more latch will be bound than in the other scenario.
These things should be worked in to the design of the next test.
Positive Control Amplicon Test 2[edit]
See above discussion. Since I do not have a lot of the beads, it will be hard to test the first hypothesis. I think the second is more important any way, and kind of encompasses the first.
Protocol[edit]
- Latch and Padlock Hybridization
- Suspend 1 uL (4 ug) beads per sample in 100 uL wash buffer (total)
- Apply magnet for 30 sec and remove supernatant
- Add 2 uL oligo per sample to bead solution (see above table); incubate at RT for 5 min
- Apply magnet for 30 sec and remove supernatant
- Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
- Mix together samples according to table in 0.2 mL tubes; use Padlock0001-68
- Dissolve target in 50 uL wash buffer and heat at 65C for 5 minutes
- Chill on ice for 3 minutes
- Add sample to prepared strep beads; vortex to suspend
- Incubate for 30 min at 37C
- Apply magnet and remove supernatant
- Add 100 uL wash buffer and vortex to suspend beads; apply magnet and remove supernatant
- Repeat wash step above
- Add 100 uL cold Low Salt Buffer and vortex to suspend; apply magnet and remove supernatant
- Resuspend with 20 uL 1x Amp Ligase buffer
- Circularization
- Prepare 20 uL KLN mix
- Prepare 4.1x master mix of Phusion mix
- Add 20 uL Phusion mix to 20 uL of reaction for samples 2-5
- Add 2 uL KLN mix to 20 uL of reaction for sample 1
- Incubate at 55C overnight; continued tomorrow
Tube | Biotin-Amplicon | uL beads added | uL biotin probe added |
1 | Amp1 | 5 | 10 |
2 | Amp3 | 5 | 10 |
Sample No | Sample Name | LatchX2 (300 ng - 2 uL 10 uM) uL | LatchX3 (300 ng - 2 uL 10 uM) uL | PCAmp1 | PCAmp3 | Expected Product |
1 | X2-1X | 2 | 0 | X | Amp1 | |
2 | X2-5X | 10 | 0 | X | Amp1 | |
3 | X2-10X | 2* | 0 | X | Amp1 | |
4 | X2-50X | 10* | 0 | X | Amp1 | |
5 | X3-1X | 0 | 2 | X | Amp3 | |
6 | X3-5X | 0 | 10 | X | Amp3 | |
7 | X3-10X | 0 | 2* | X | Amp3 | |
8 | X3-50X | 0 | 10* | X | Amp3 | |
9 | X2Flip | 2 | 0 | X | None | |
10 | X3 Flip | 0 | 2 | X | None |