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

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>Djacobse
>Djacobse
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=C Probe SOD1/VIM Test=
=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.
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.


<gallery widths=300px heights=200px perrow=2 caption="BLAST results for probe target regions against the mouse genome and transcriptome">
<gallery widths=300px heights=200px perrow=2 caption="BLAST results for probe target regions against the mouse genome and transcriptome">
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File:SOD1-PB.png|SOD1 PB probe
File:SOD1-PB.png|SOD1 PB probe
</gallery>
</gallery>
==Protocol==
<ol>
<li>C-Probe Hybridization</li>
<ol type="A">
<li>Split cells into 2 samples (1 mL methanol each)</li>
<li>Pellet cells via centrifugation at 600g for 3 min</li>
<li>Put 1 uL C1 Probe and 1 uL C2 probe into a 0.2 mL tube</li>
<li>Heat C probes to 90C for 5 min; Chill probes on ice to quench</li>
<li>Add probes to CPH buffer for final concentration of 100 nM (Add 1 uL probe mixture to 0.5mL hybridization buffer)</li>
<li>Incubate probes at 40C for 1 hour with vigorous agitation</li>
<li>Wash three times with 200 uL wash buffer and pelleting at 600xg for 3 min</li>
<li>Incubate cells for 20 min at 40C in 200 uL stringent wash buffer</li>
</ol>
<li>Latch and Padlock Hybridization</li>
<ol type="A">
<li>Pellet at 600xg for 3 min and remove supernatant</li>
<li>Resuspend pellet in 100 uL LPH buffer per sample (600 uL S1, 200 uL S2)</li>
<li> Add 1 uL of each insert/backbone (final conc. 100 nM each)</li>

Revision as of 20:11, 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. 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

    1. C-Probe Hybridization
      1. Split cells into 2 samples (1 mL methanol each)
      2. Pellet cells via centrifugation at 600g for 3 min
      3. Put 1 uL C1 Probe and 1 uL C2 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 1 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 per sample (600 uL S1, 200 uL S2)
      3. Add 1 uL of each insert/backbone (final conc. 100 nM each)