Daniel:Notebook/ComboLock/2016-7-7
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Sequential Hybridization Test (Started 7-5-2016)
Protocol
- 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
Source | ng/uL |
C Probe-Seq Tube 1 | 243.1 |
C Probe-Seq Tube 2 | 287.9 |
Library Prep
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
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
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 |
Protocol
- 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