Daniel:Notebook/ComboLock/2016-8-25
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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
- 2016-08-20-PosConTest-SizeSelect.png
Before Image
- 2016-08-20-PosConTest-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 | 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 |