Daniel:Notebook/ComboLock/2016-6-29: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Djacobse
No edit summary
>Djacobse
No edit summary
 
(One intermediate revision by the same user not shown)
Line 282: Line 282:
</ol>
</ol>


[[Category:ComboLock]] [[Category:20160628|Ab Test]] [[Category:20160629|SOD1-VIM Test]]
[[Category:ComboLock]] [[Category:20160628]] [[Category:20160629]]

Latest revision as of 22:26, 6 July 2016

Antibody Stage 2 Test 2 (Started yesterday)[edit]

Back to Calendar

Protcol[edit]

  1. Latch and Padlock Hybridization
    1. Heat inactivate enzyme by incubating for 2 minutes at 94C
  2. Exonuclease Digestion
    1. Prepare exonuclease I/III mix by mixing 15 uL exonuclease I (20 units/μL) and 3 uL exonuclease III (100 units/μL) in 1:1 ratio
    2. Add 2 μL of exonuclease I/III mix to reaction
    3. Mix the reaction by swirling pipette around the well 5 times
    4. Incubate reaction at 37 ºC for 2 hours
    5. Heat inactivate enzyme by incubating at 94C for 5 minutes
  3. qPCR
    1. Set up reaction according to table
    2. Sample Lane Batch AmpR Index Cells 2X Kapa SYBR qPCR MM 100 uM AmpF 6.4 10 uM AmpR6.3-IndXX H2O Total Volume (uL)
      Sample 1 A1 B1 1 2 25 0.5 5 17.5 50
      Sample 2 A2 B2 2 2 25 0.5 5 17.5 50
      Sample 3 A3 B3 3 2 25 0.5 5 17.5 50
      Sample 4 A4 B4 4 2 25 0.5 5 17.5 50
      Sample 5 A5 B5 5 2 25 0.5 5 17.5 50
      Sample 6 A6 B6 6 2 25 0.5 5 17.5 50
      Sample 7 A7 B7 7 2 25 0.5 5 17.5 50
    3. Master Mix
      1. 165 uL KAPA SyberFast MM
      2. 115.5 uL nfH2O
      3. 3.3 uL AmpF 6.4
    4. Add 43 uL Master Mix to each well
    5. qPCR Cycles
      1. 95C 3 min
      2. 95C 3 sec
      3. 55C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x20
      7. 72C 2 min
      8. 16C hold

    qPCR Results[edit]

    The results are confusing since even the negative control samples amplified. I need to repeat the experiment.

    C Probe SOD1/VIM Test[edit]

    On Andrew's advice I'm also going to do another test using mouse cells (and human cells), this time with C-probes. To test more than one variable, I'm also going to use SOD1 and VIM. Below I've included the BLAST results testing the human probes against the mouse genome and transcriptome. VIM PA actually has a near-perfect match, but VIM PB does not match anywhere near VIM PA, so both probes should be unable to bind.

    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 100 10 uL
    nf H2O NA NA NA 690 uL

    LPH Buffer

    Reagent Stock Final Dilution Amt in 100 uL (uL)
    Oligos 10 uM 100 nM 100 1 uL
    SSC 20X 1X 5 20 uL
    Rnasin 40000 U/mL 40 U/mL 100 1 uL
    PBS 10X 1X 10 10 uL
    nf H2O NA NA NA 68 uL

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


    Experiment[edit]

    1. C-Probe Hybridization
      1. Using same cells as yesterday 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. Incubate probes at 40C for 1 hour with vigorous agitation
      7. Wash three times with 200 uL wash buffer and pelleting at 600xg for 3 min
      8. 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