Daniel:Notebook/HiResChrPaint/2014-9-10
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Padlock Probes II[edit]
Nanodrop Results[edit]
Sample | 1:1 ng/uL | 5:1 ng/uL | 5:1 ng/uL Projected | 10:1 ng/uL | 10:1 ng/uL Projected | Average ng/uL | Standard Deviation | ug in 100 uL |
PPRL-A | 1944.1 | 373 | 1865 | 184.4 | 1844 | 1884.4 | 52.8 | 188.4 |
PPRL-B | 2064.8 | 421.7 | 2108.5 | 203 | 2030 | 2067.8 | 39.3 | 206.8 |
Usually after ethanol precipitation I get really high results, so I'll just say these are fine and continue as though I have a reasonable amount of material.
DpnII/USER Digestion[edit]
Reaction Table
Reagent | PPRL-A1 (uL) | PPRL-A2 (uL) | PPRL-B1 (uL) | PPRL-B2 (uL) |
amplicon | 50 | 50 | 50 | 50 |
ug Input | 94.2 | 94.2 | 103.4 | 103.4 |
USER | 5 | 5 | 5 | 5 |
nf H2O | 30 | 30 | 30 | 30 |
Total | 85 | 85 | 85 | 85 |
- Set up reaction according to table in a 0.2 mL PCR tube
- Incubate for 2 hours at 37C
- Add 10 uL DpnII buffer (10X)
- Incubate at 94C for 2 minutes
- Cool to 37C and incubate for 3 minutes
- Add 5 uL DpnII and incubate at 37C for 2 hours
- Heat inactivate DpnII at 65C for 20 minutes
- [[Daniel:Protocols/
Nanodrop Results[edit]
Sample | PPRL-A1 | PPRL-A2 | PPRL-B1 | PPRL-B2 |
ng/uL | 154.1 | 131 | 153 | 163.7 |
ug in 40 uL | 6.2 | 5.2 | 6.1 | 6.5 |
ug total | 24.1 |
TBE Gel[edit]
- For each sample, 5 uL TBE, 2 uL 6x dye, 1 uL sample; Load 7.5 uL each
- Run gel at 220V for 25 minutes
Lane | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
Sample | Low Mass Ladder | PPRL-A, pre-digest 10:1 | PPRL-A, pre-digest 40:1 | PPRL-A1 | PPRL-A2 | PPRL-B, pre-digest 10:1 | PPRL-B, pre-digest 40:1 | PPRL-B1 | PPRL-B2 |
Gel Image