Daniel:Notebook/ComboLock/2016-10-28
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Beads UMI Test
Protocol
- Circularization
- Heat inactivate enzyme by incubating for 2 minutes at 94C
- Exonuclease Digestion
- Prepare exonuclease I/III mix by mixing 20 uL exonuclease I (20 units/μL) and 4 uL exonuclease III (100 units/μL) in 1:1 ratio
- Add 2 μL of exonuclease I/III mix to reaction
- Mix the reaction by swirling pipette around the well 5 times
- Incubate reaction at 37 ºC for 2 hours
- Heat inactivate enzyme by incubating at 94C for 5 minutes
- qPCR
- Set up reaction according to table in the next section
- Make master mixes according to following recipes
- 338.1 uL nfH2O
- 402.5 uL SYBR Fast
- 16.1 uL 10 uM AmpF
- Add 43 uL master mix to each well
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x24
- 72C 2 min
- 16C hold
- TBE Gel
- Mix 160 uL TBE, 40 uL 6x loading dye
- Aliquot 10 uL per sample/ladder lane onto parafilm
- Add 2 uL of sample or ladder to correct drop
- Load 10 uL in to well
- Run gel for 22 minutes at 250V
- Open gel and stain with 3 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc
qPCR Sample Table
Sample | Latch | Lane | AmpR Index | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 10 uM Forward Primer | 10 uM Reverse Primer | H2O | Total Volume (uL) |
Sample 1A | Latch0001 | A1 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1B | Latch0002 | B1 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1C | LatchX4 | C1 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2A | Latch0001 | D1 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2B | Latch0002 | E1 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2C | LatchX4 | F1 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3A | Latch0001 | G1 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3B | Latch0002 | H1 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3C | LatchX4 | A12 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4A | Latch0001 | B12 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4B | Latch0002 | C12 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4C | LatchX4 | D12 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 5A | Latch0001 | E12 | 24 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 5B | Latch0002 | F12 | 24 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 5C | LatchX4 | G12 | 24 | 2 | 25 | 1 | 1 | 21 | 50 |
NTC | NA | H12 | 25 | 2 | 25 | 1 | 1 | 21 | 50 |
Results
- 2016-10-06-qPCR-PCCTest1.png
qPCR curve
- 2016-10-06-PCCTest1-AmpFAmpR.png
Gel image