Daniel:Notebook/ComboLock/2016-7-1

From ZhangLabWiki
Jump to navigation Jump to search

Back to Calendar

Antibody Stage 2 Test 2 (Started 6-28-2016)[edit]

Continuing from 6-30

  1. Ethanol Precipitation
    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]

Batch Source ng/uL
Ab1 15.5
Ab2 1.0

C Probe SOD1/VIM Test (Started 6-29-2016)[edit]

  1. Size Selection
    1. Take 20 uL sample from each of the first 4 lanes
    2. Add 4 uL 6X loading dye to each
    3. Aliquot 42 uL each into 4 lanes
    4. 4 uL ladder with 4 uL gel loading dye and 16 uL TBE buffer
    5. Run gel for 25 min at 220V
    6. Stain with 3 uL SYBR Gold for 3 min
    7. Image in gel doc; cut out selected bands and place in 0.5 mL tube with hole in the bottom inside a 1.5 mL tube
    8. Centrifuge gel at 14000rpm for 1.5 minutes
    9. Remove 0.5 mL tube and add 500 uL TE buffer to shredded gel
    10. Incubate at 37C for at least an hour with vigorous shaking
    11. Centrifuge at 14000rpm for 1.5 minutes
    12. Transfer supernatant to a nanosep column
    13. Centrifuge at 14000rpm for 1.5 minutes
    14. Transfer flow through to a 2 mL tube
  2. Ethanol Precipitation
    1. Add 1.25 mL 100% EtOH, 50 uL 3M NaOAc, and 2 uL glycoblue to sample
    2. Incubate two hours at -80C
    3. Centrifuge at 4 C for 25 minutes at 14000 rpm
    4. Remove supernatant and add 750 uL chilled 75% EtOH
    5. Centrifuge for 15 minutes at minutes at 4 C at 14000 rpm
    6. Remove supernatant and dry the pellet in the hood
    7. Resuspend pellet in 20 uL volume nfH20
    8. Measure in Nanodrop
    9. Store DNA at 4C

    Nanodrop Results[edit]

    Sample ng/uL
    Hs-Sample 2 38.5
    mm-Sample 4 43.3

    C Probe SOD1/VIM Test 2[edit]

    The results from the first SOD1/VIM test were ambiguous, since the wrong samples amplified (although it was the right NUMBER of samples). So, besides sequencing the results (see above for prep), I'm also just going to run the experiment again.

    Protocol[edit]

    Buffer Prep[edit]

    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

    Resuspended SOD1 C probes in 40 uL nfH2O each (100 uM)

    Experiment[edit]

    1. C-Probe Hybridization
      1. Using same cells as 6-28 cells to a new tube such that there are 100000 cells present
        1. Human-256 uL; Mouse-85 uL
      2. Pellet cells via centrifugation at 600g for 3 min
      3. Combine 1.5 uL of each C probe into a 0.2 mL tube
      4. Heat C probes to 90C for 5 min; Chill probes on ice to quench
      5. Add probes to CPH buffer for final concentration of 100 nM (Add 2 uL probe mixture to 0.5mL hybridization buffer)
      6.   C Probes-VIM (C1+C2) C Probes-SOD1 (C1+C2) U87MG 3T3 Cells
        Sample 1 X X X  
        Sample 2     X  
        Sample 3 X X   X
        Sample 4       X
        Sample 5 X X    
      7. Incubate probes at 40C for 1 hour with vigorous agitation
      8. Wash three times with 200 uL wash buffer and pelleting at 600xg for 3 min
      9. 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; use Latch0001 and Padlock0001-68
      3. Incubate for 30 min at 37C following the sample matrix
      4. Wash twice with 200 uL wash buffer
        1. Wash by pelleting cells at 600xg for 3 min
      5. 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