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

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=C Probe SOD1/VIM Test=
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.
<gallery widths=300px heights=200px perrrow=2 caption="BLAST results for probe target regions against the mouse genome and transcriptome">
File:VIM_PA.png|VIM PA probe
File:VIM_PB.png|VIM PB probe
File:SOD1-PA.png|SOD1 PA probe
File:SOD1-PB.png|SOD1 PB probe
</gallery>

Revision as of 20:08, 29 June 2016

Antibody Stage 2 Test 2 (Started yesterday)

Back to Calendar

Protcol

  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

    C Probe SOD1/VIM Test

    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.