Daniel:Notebook/ComboLock/2016-7-7
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Sequential Hybridization Test (Started 7-5-2016)[edit]
Protocol[edit]
- Ethanol precipitation (from yesterday)
- Centrifuge at 4 C for 25 minutes at 14000 rpm
- Remove supernatant and add 750 uL chilled 75% EtOH
- Centrifuge for 15 minutes at minutes at 4 C at 14000 rpm
- Remove supernatant and dry the pellet in the hood
- Resuspend pellet in 20 uL volume nfH20
- Measure in Nanodrop
- Store DNA at 4C
Nanodrop Results[edit]
Source | ng/uL |
C Probe-Seq Tube 1 | 243.1 |
C Probe-Seq Tube 2 | 287.9 |
Library Prep[edit]
Mixed samples according to table.
Sample | ng/uL | uL added | final mass (ug) |
C Probe-Seq Tube 1 | 243.1 | 15 | 3.6 |
C Probe-Seq Tube 2 | 287.9 | 15 | 4.3 |
Sample Name: DEJ-CL-C03
C Probe/Antibody Mix[edit]
I think it is time to try an experiment with the C-Probes and antibodies. Just to get sequence information, if we can. I think I will stick to the sequential hybridization method for now.
Buffer Prep[edit]
Prepare 1 mL of hybridization buffer and aliquot in 100 uL fractions. Freeze unused.
Reagent | Stock | Final | Dilution | Amt in 100 uL (uL) | Amt in 1 mL (uL) |
Oligos | 100 uM | 100 nM | 1000 | 1 uL | 0 |
Antibodies | 1500 nM | 0.5 nM | 3000 | 1 uL | 0 |
BSA | 50 mg/mL (5%) | 0.10% | 500 | 0.5 | 2 |
RNAsin | 40000 U/mL | 40 U/mL | 1000 | 0.1 | 1 |
EDTA | 500 mM | 5 mM | 100 | 1 | 10 |
Salmon Sperm DNA | 10 mg/mL | 0.1 mg/mL | 100 | 1 uL | 10 |
PBS | 10X | 1X | 10 | 10 uL | 100 |
nf H2O | NA | NA | NA | 85 uL | 877 |
Stringent Wash Buffer
Reagent | Stock | Final | Dilution | Amt in 1 mL (uL) |
PBS | 10X | 1X | 10 | 100 uL |
SSC | 20X | 4X | 5 | 200 uL |
Rnasin | 40000 U/mL | 40 U/mL | 1000 | 1 uL |
nf H2O | NA | NA | NA | 700 uL |
LPH Buffer
Reagent | Stock | Final | Dilution | Amt in 1 mL (uL) |
Oligos | 100 uM | 100 nM | 1000 | 1 uL |
SSC | 20X | 1X | 20 | 50 uL |
Rnasin | 40000 U/mL | 40 U/mL | 1000 | 1 uL |
PBS | 10X | 1X | 10 | 100 uL |
nf H2O | NA | NA | NA | 850 uL |
Protocol[edit]
- Hybridization
- Measure cell concentration using 10 uL cell sample in 90 uL nfH2O
- Human- cells/mL
- Extract cells to a new tube such that there are 100000 cells present
- Human- 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
- Resuspend cells in 100 uL hybridization buffer
- C Probe preparation
- Mix all probes, 1 uL of each 100 uM probe (4 uL total VIM+SOD1 C1 and C2)
- Add 6 uL nfH2O to bring concentration to 10 uM for each probe
- Incubate at 90C for 5 min; chill on ice to quench
- Antibody Prep
- Add 1 uL of VIM-PA and VIM-PB each to 0.2 mL tube
- Add 28 uL nfH2O (reduces concentration to ~50 nM for each)
- Add 1 uL each probe mix according to sample matrix
- Incubate probes at 40C for 1 hour with vigorous agitation
- Wash three times with 200 uL wash buffer and pelleting at 600xg for 3 min
- Incubate cells for 20 min at 40C in 200 uL stringent wash buffer
- Latch and Padlock Hybridization
- Pellet at 600xg for 3 min and remove supernatant
- Resuspend pellet in 100 uL LPH buffer with 100 nM Latch0001
- Incubate for 30 min at 37C
- Wash twice with 200 uL wash buffer
- Wash by pelleting cells at 600xg for 3 min
- Resuspend pellet in 100 uL LPH buffer with 100 nM Padlock0001-68
- Incubate for 30 min at 37C
- 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 (XX hours)
- Continued tomorrow
C Probes (1 uL each) | Antibodies (1 uL each) | U87MG | 3T3 Cells | |
Sample 1 | X | X | X | |
Sample 2 | X | |||
Sample 3 | X | X | X | |
Sample 4 | X | |||
Sample 5 | X | X |