Daniel:Notebook/ComboLock/2016-7-7

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Revision as of 18:14, 7 July 2016 by >Djacobse (→‎Library Prep)
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Sequential Hybridization Test (Started 7-5-2016)

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Protocol

  1. Ethanol precipitation (from yesterday)
    1. Centrifuge at 4 C for 25 minutes at 14000 rpm
    2. Remove supernatant and add 750 uL chilled 75% EtOH
    3. Centrifuge for 15 minutes at minutes at 4 C at 14000 rpm
    4. Remove supernatant and dry the pellet in the hood
    5. Resuspend pellet in 20 uL volume nfH20
    6. Measure in Nanodrop
    7. 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

  1. Hybridization
    1. Measure cell concentration using 10 uL cell sample in 90 uL nfH2O
      1. Human- cells/mL
    2. Extract cells to a new tube such that there are 100000 cells present
      1. Human- uL; Mouse-85 uL
    3. Pellet cells via centrifugation at 600g for 3 min
    4. Add 200 uL Wash Buffer (0.05% Tween 20 in 1X PBS)
    5. Pellet cells via centrifugation at 600g for 3 min