Daniel:Notebook/ComboLock/2016-8-25
Positive Control Amplicon Test 2 (Started Monday)
I'm going to run library prep on the product from qPCR from Tuesday. I'll take the two "flip" lanes (wrong latch for the amplicon template) because I want to see what is going on. I'll also take the best results from X2 and X3.
Protocol-Library Prep
- Size Selection
- Add 20 uL sample lanes 1 and 8-10, 5 uL 6X loading dye and 5 uL TBE to sample lanes
- Lane 1: X2-1X
- Lane 8: X3-50X
- Lane 9: X2-Flip
- Lane 10:X3-Flip
- Aliquot 30 uL each into 4 sample lanes
- 2 uL ladder with 5 uL gel loading dye and 28 uL TBE buffer
- Run gel for 20 min at 250V
- Stain with 3 uL SYBR Gold for 3 min
- Image in gel doc; cut out selected bands and place in 0.5 mL tube with hole in the bottom inside a 1.5 mL tube
- 2016-08-25-PosConAmpTest2-SizeSelect.png
Before Image
- 2016-08-25-PosConAmpTest2-SizeSelect-After.png
After Image
- Centrifuge gel at 14000rpm for 1.5 minutes
- Remove 0.5 mL tube and add 500 uL TE buffer to shredded gel
- Incubate at 37C for at least an hour with vigorous shaking
- Centrifuge at 14000rpm for 1.5 minutes
- Transfer supernatant to a nanosep column
- Centrifuge at 14000rpm for 1.5 minutes
- Transfer flow through to a 2 mL tube
- Ethanol Precipitation
- Add 1.25 mL 100% EtOH, 50 uL 3M NaOAc, and 2 uL glycoblue to sample
- Incubate overnight at -80C; continued tomorrow
Positive Control Amplicon Test
Preliminary Sequencing Results
From looking at the results from the four sample index lanes. Each of the indexes corresponds to an experiment, I've attached the expected table below.
Sample No | Sample Name | Index | Expected Product |
1 | Amp1 Test | 10 | Amp1 |
2 | Amp3 Test | 11 | Amp3 |
3 | LatchX2 Specificity | 12 | Amp1 |
4 | LatchX3 Specificity | 13 | Amp3 |
Also below is the gel image from size selection. Note that index 11 didn't have any product of the right size, so I don't expect any results from that column.
- 2016-08-20-PosConTest-SizeSelect.png
Before Image
- 2016-08-20-PosConTest-SizeSelect-After.png
After Image
Now we can look at the results. In order to get these I used grep on the barcodes that are specific to each of the three positive control pieces (C1, C2 and C3). I grepped each barcode alone and in pairs with each other. Note that C1-C2 and C3-C2 are the expected products, C1-C3 should never exist.
Name | Barcode Sequence | Index 10 | Index 11 | Index 12 | Index 13 |
Latch6 BC (C3) | GTTGGCAG | 4 | 95 | 7658 | 11266 |
Latch8 BC RC (C1) | TACAAATA | 1140 | 187 | 14963 | 26222 |
Latch9 BC RC (C2) | GTAGGCGC | 1069 | 77 | 22059 | 37200 |
Intersect L8-L9 (C1-C2) | TACAAATA,GTAGGCGC | 1025 | 0 | 14047 | 25016 |
Intersect (C3-C2) | GTTGGCAG,GTAGGCGC | 0 | 2 | 7163 | 10759 |
Intersect (C1-C3) | GTTGGCAG,TACAAATA | 0 | 0 | 0 | 0 |
Percent C2 Reads from Amplicons | 95.884 | 2.597 | 96.151 | 96.169 | |
Perecent On Target | 100 | 0 | 66.2 | 30.1 |
The results are quite encouraging, as in the lane where only C1-C2 (PCAmp1) was added, only C1-C2 shows up. In the two mixed lanes, however, we get reads from both fragments. Because of this, I am investigating further. The "Flip" samples from Positive Control Amplicon Test 2 test the wrong latch-template combination without the other latch or template (e.g. tests LatchX3 and C1-C2). Therefore, sequencing them will give me a better idea of what is going on. I think that the latch is able to bind even when it is wrong. This is because the 18bp sequences flanking each latch are the same (they are the adapter sequences). If that is the case, I should see sequencing results that match the latch, not the padlock.
One additional note. Extracting only the barcodes is not proof enough that the barcodes are in the right place. So I manually aligned three reads. They are all perfect.