Daniel:Notebook/ComboLock/2016-6-28
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Antibody Stage 2 Test (Started yesterday)[edit]
Protocol[edit]
- Latch and Padlock Hybridization
- Heat inactivate enzyme by incubating for 2 minutes at 94C
- Exonuclease Digestion
- Prepare exonuclease I/III mix by mixing 2 uL exonuclease I (20 units/μL) and 10 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
- Master Mix
- 165 uL KAPA SyberFast MM
- 115.5 uL nfH2O
- 3.3 uL AmpF 6.4
- 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 x20
- 72C 2 min
- 16C hold
- qPCR
- Set up reaction according to table
- Master Mix
- 192.5 uL KAPA SyberFast MM
- 134.75 uL nfH2O
- 3.85 uL AmpF 6.4
- 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 x20
- 72C 2 min
- 16C hold
- VIM-Ab-LPTest2 Results 20160628.png
qPCR Results
- 2016-06-28-CombolockAb-LP-Test2.jpg
Gel Results
- Hybridization
- Measure cell concentration using 10 uL cell sample in 90 uL nfH2O
- Human-3.9E5 cells/mL; Mouse-1.2E6 cells/mL
- Extract cells to a new tube such that there are 100000 cells present
- Human-256 uL; Mouse-85 uL
- Pellet cells via centrifugation at 600g for 3 min
- Add 200 uL Wash Buffer (0.05% Tween 20 in 1X PBS)
- Pellet cells via centrifugation at 600g for 3 min
- Add PLA mix and probes to create hybridization solution
- Resuspend cells in 10 uL PBS
- Mix 1 uL cell lysate with 3 uL Probe mix (10000 cells/reaction)
- Incubate 1.5 hours at RT
- Add 200 uL 0.05% Tween 20 in PBS
- Wash twice with 0.05% Tween 20 in PBS
- Pellet cells at 600xg for 3 min
- Latch and Padlock Hybridization
- Resuspend pellet in 100 uL LPH buffer per sample (600 uL S1, 200 uL S2)
- Add 1 uL of each insert/backbone (final conc. 100 nM each)
- Incubate for 30 min at 37C following the sample matrix
- Wash twice with 200 uL wash buffer
- Wash by pelleting cells at 600xg for 3 min
- Resuspend with 20 uL 1x Amp Ligase buffer
- Circularization
- Prepare 20 uL KLN mix
- Add 2 uL KLN mix to 20 uL of reaction
- Incubate at 55C overnight (16 hours)
- Continued tomorrow
- Latch-Padlock Hybridization (LPH) Buffer
- 100 nM insert/backbone oligos
- 1X SSC
- 40 U/mL RNasin
- PBS
- KLN Mix 20 uL total
- 4 uL HemoKlentaq (20% v/v)
- 2 μL Ampligase (0.5 U/uL)
- 2 uL 1 mM dNTP mix (100 μM ea)
- Add 10 uL 10mM dNTP mix to 90 uL nfH2O
- 2 uL 1x Ampligase Buffer 10x
- 10 uL nfH2O
Sample | Lane | Batch | AmpR Index | Cells | 2X Kapa SYBR qPCR MM | 100 uM AmpF 6.4 | 10 uM AmpR6.3-IndXX | H2O | Total Volume (uL) |
Sample 1 | A1 | B1 | 1 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 2 | A2 | B2 | 2 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 3 | A3 | B3 | 3 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 4 | A4 | B4 | 4 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 5 | A5 | B5 | 5 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 6 | A6 | B6 | 6 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
qPCR Results[edit]
File:VIM-Ab-LPTest Results 20160628.png
qPCR Repeat[edit]
Sample | Lane | Batch | AmpR Index | Cells | 2X Kapa SYBR qPCR MM | 100 uM AmpF 6.4 | 10 uM AmpR6.3-IndXX | H2O | Total Volume (uL) |
Sample 1 | A1 | B1 | 1 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 2 | A2 | B2 | 2 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 3 | A3 | B3 | 3 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 4 | A4 | B4 | 4 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 5 | A5 | B5 | 5 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 6 | A6 | B6 | 6 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 7 | A7 | NA | 7 | 0 | 25 | 0.5 | 5 | 19.5 | 50 |
Results[edit]
The second qPCR confirmed that I am seeing a good deal of signal in many of the lanes that shouldn't have any.
Trial 2[edit]
After talking with Andrew, I want to try yesterday's experiment again with a fixed number of cells (10000). This is to prevent any clogging that may occur during the protocol if there are too many cells.
uL PA Mix | uL PB Mix | uL PLA buffer | |
PLA Mix E | 1 | 1 | 4 |
PLA Mix NC | 0 | 0 | 6 |
uL PLA Mix E | uL PLA Mix NC | uL U87MG Cells | uL 3T3 Cells | |
Sample 1 | 3 | 0 | 1 | 0 |
Sample 2 | 3 | 0 | 0 | 1 |
Sample 3 | 0 | 3 | 1 | 0 |
Sample 4 | 0 | 3 | 0 | 1 |
Sample 5 | 3 | 1 | 0 | 0 |
Batch | Cell Sample | Latch0001 (1 uL) | Padlock0001-68 (1 uL) | Padlock0001-88 (1 uL) |
Batch 1 | S1 | X | X | |
Batch 2 | S1 | X | X | |
Batch 3 | S2 | X | X | |
Batch 4 | S2 | X | X | |
Batch 5 | S3 | X | X | |
Batch 6 | S4 | X | X | |
Batch 7 | S5 | X | X |
Buffers[edit]
LPH Buffer
Reagent | Stock | Final | Dilution | Amt in 100 uL (uL) |
Oligos | 10 uM | 100 nM | 100 | 1 uL |
SSC | 20X | 1X | 5 | 20 uL |
Rnasin | 40000 U/mL | 40 U/mL | 100 | 1 uL |
PBS | 10X | 1X | 10 | 10 uL |
nf H2O | NA | NA | NA | 68 uL |