Daniel:Notebook/ComboLock/2016-7-7

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Sequential Hybridization Test (Started 7-5-2016)[edit]

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Protocol[edit]

  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[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]

  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
    6. Resuspend cells in 100 uL hybridization buffer
    7. C Probe preparation
      1. Mix all probes, 1 uL of each 100 uM probe (4 uL total VIM+SOD1 C1 and C2)
      2. Add 6 uL nfH2O to bring concentration to 10 uM for each probe
      3. Incubate at 90C for 5 min; chill on ice to quench
    8. Antibody Prep
      1. Add 1 uL of VIM-PA and VIM-PB each to 0.2 mL tube
      2. Add 28 uL nfH2O (reduces concentration to ~50 nM for each)
    9. Add 1 uL each probe mix according to sample matrix
    10.   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    
    11. Incubate probes at 40C for 1 hour with vigorous agitation
    12. Wash three times with 200 uL wash buffer and pelleting at 600xg for 3 min
    13. Incubate cells for 20 min at 40C in 200 uL stringent wash buffer
  2. Latch and Padlock Hybridization
    1. Pellet at 600xg for 3 min and remove supernatant
    2. Resuspend pellet in 100 uL LPH buffer with 100 nM Latch0001
    3. Incubate for 30 min at 37C
    4. Wash twice with 200 uL wash buffer
      1. Wash by pelleting cells at 600xg for 3 min
    5. Resuspend pellet in 100 uL LPH buffer with 100 nM Padlock0001-68
    6. Incubate for 30 min at 37C
    7. Wash twice with 200 uL wash buffer
      1. Wash by pelleting cells at 600xg for 3 min
    8. Resuspend with 20 uL 1x Amp Ligase buffer
  3. Circularization
    1. Prepare 20 uL KLN mix
    2. Add 2 uL KLN mix to 20 uL of reaction
    3. Incubate at 55C overnight (XX hours)
    4. Continued tomorrow