Daniel:Notebook/ComboLock/2016-11-7: Difference between revisions

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(Created page with "=Cell Test with RCA= Back to Calendar This experiment is designed to see if I can get mRNA targeting C-Probes to work in cells, this time with ...")
 
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==Protocol==
==Protocol==


<ol>
<li>Cell Preparation</li>
<ol type="A">
<li>Pellet cells via centrifugation at 600g for 3 min</li>
<li>Pellet cells via centrifugation at 600g for 3 min</li>
<li>Wash with 200 uL wash buffer and pellet cells via centrifugation at 600g for 3 min</li>
<li>Wash with 200 uL wash buffer and pellet cells via centrifugation at 600g for 3 min</li>

Revision as of 17:44, 7 November 2016

Cell Test with RCA

Back to Calendar

This experiment is designed to see if I can get mRNA targeting C-Probes to work in cells, this time with RCA. I'll be trying both VIM and SOD1, just to have two separate genes.

Protocol

  1. Cell Preparation
    1. Pellet cells via centrifugation at 600g for 3 min
    2. Wash with 200 uL wash buffer and pellet cells via centrifugation at 600g for 3 min
    3. Add 100 uL 50% hematoxylin (filtered) and pellet cells via centrifugation at 600g for 3 min
    4. Wash once with 200 uL wash buffer and pellet cells via centrifugation at 600g for 3 min
    5. Wash 2 times with 200 uL wash buffer prime and pellet cells via centrifugation at 600g for 3 min
    6. Pellet cells via centrifugation at 600g for 3 min
    7. Combine 0.5 uL of each C probe into a 0.2 mL tube with 2 uL nfH2O
    8. Heat C probes to 90C for 5 min; Chill probes on ice to quench
    9. Add probes to CPH buffer according to sample matrix, 1 uL probe mix per sample
    10. Incubate probes at 40C for 1 hour with vigorous agitation
    11. Wash three times with 200 uL wash buffer and pelleting at 600xg for 3 min
    12. 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 Padlock0201-68 and 100 nM Latch0001
    3. Incubate for 30 min at 37C
    4. Wash twice with 100 uL wash buffer by pelleting cells at 600xg for 3 min
    5. Incubate cells for 20 min at 40C in 150 uL stringent wash buffer
    6. Resuspend with 20 uL 1x Amp Ligase buffer and transfer to a 0.2 mL tube
  3. Circularization
    1. Prepare 20 uL KLN mix
    2. Add 2 uL KLN mix to 20 uL of reaction for sample 1
    3. Incubate at 55C overnight (XX hours)
    4. Continued tomorrow