Daniel:Notebook/ComboLock/2016-6-23: Difference between revisions
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<li>Put 1 uL C1 Probe and 1 uL C2 probe into a 0.2 mL tube</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>Heat C probes to 90C for 5 min; Chill probes on ice to quench</li> | ||
<li>Add probes to | <li>Add probes to CP 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>Incubate probes at 40C for 1 hour with vigorous agitation</li> | ||
<li>Wash three times with wash buffer and pelleting at 600xg for 3 min</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 stringent wash buffer</li> | <li>Incubate cells for 20 min at 40C in 200 uL stringent wash buffer</li> | ||
</ol> | </ol> | ||
<li>Latch and Padlock Hybridization</li> | <li>Latch and Padlock Hybridization</li> | ||
<ol type="A"> | <ol type="A"> | ||
<li>Pellet at 600xg for 3 min</li> | <li>Pellet at 600xg for 3 min and remove supernatant</li> | ||
<li> | <li>Resuspend pellet in 50 uL hybridization buffer with 100 nM insert/backbone oligos for 30 min at 37C</li> | ||
<li>Wash twice</li> | <li>Wash twice</li> | ||
<ol type="a"><li>Wash by pelleting cells at 600xg for 3 min</li></ol></ol> | <ol type="a"><li>Wash by pelleting cells at 600xg for 3 min</li></ol></ol> | ||
Line 80: | Line 80: | ||
</ol> | </ol> | ||
</ol> | </ol> | ||
</ol> | |||
===Buffers=== | |||
<ol type="A"> | |||
<li>C-Probe (CP) Buffer</li> | |||
<ol type="a"> | |||
<li>100 nM insert/backbone oligos</li> | |||
<li>1X SSC</li> | |||
<li>40 U/mL RNasin</li> | |||
<li>PBS</li> | |||
</ol> | |||
<li>Wash Buffer</li> | |||
<ol type="a"> | |||
<li>PBS</li> | |||
<li>0.1% Tween</li> | |||
<li>4 U/mL RNasin</li> | |||
</ol> | </ol> |
Revision as of 22:50, 22 June 2016
Stage 2 Test
The purpose of this experiment is to test the latch and padlock binding, termed stage 2 (at the moment). This is based on the success of the VIM binding for both C-Probes and antibodies.
Protocol
Sample Matrix
Sample | VIM-C1 (1 uL) | VIM-C2 (1 uL) |
Sample 1 | X | X |
Sample 2 |
- C-Probe Hybridization
- Split cells into 2 samples (1 mL methanol each)
- Pellet cells via centrifugation at 600g for 3 min
- Put 1 uL C1 Probe and 1 uL C2 probe into a 0.2 mL tube
- Heat C probes to 90C for 5 min; Chill probes on ice to quench
- Add probes to CP buffer for final concentration of 100 nM (Add 1 uL probe mixture to 0.5mL hybridization buffer)
- Incubate probes at 40C for 1 hour with vigorous agitation
- Wash three times with 200 uL wash buffer and pelleting at 600xg for 3 min
- Incubate cells for 20 min at 40C in 200 uL stringent wash buffer
- Latch and Padlock Hybridization
- Pellet at 600xg for 3 min and remove supernatant
- Resuspend pellet in 50 uL hybridization buffer with 100 nM insert/backbone oligos for 30 min at 37C
- Wash twice
- Wash by pelleting cells at 600xg for 3 min
- Circularization
- Prepare KLN mix with:
- 20% v/v HemoKlentaq
- 0.5 U/μL Ampligase
- 100 μM of dNTP mix
- 1x Ampligase Buffer
- Add 2 uL KLN mix to 20 uL of reaction
- Incubate at 55C for 4-20 hours
- Heat inactivate enzyme by incubating for 2 minutes at 94C
- Exonuclease Digestion
- Prepare exonuclease I/III mix by mixing exonuclease I (20 units/μL) and exonuclease III (200 units/μL) in 1:1 ratio
- Add 2 μL of exonuclease I/III mix to reaction
- Mix the reaction by swirling pipette around the well 5 times
- Incubate reaction at 37 ºC for 2 hours
- Heat inactivate enzyme by incubating at 94C for 5 minutes
- qPCR
- Set up reaction according to table
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x20
- 72C 2 min
- 16C hold
Buffers
- C-Probe (CP) Buffer
- 100 nM insert/backbone oligos
- 1X SSC
- 40 U/mL RNasin
- PBS
- Wash Buffer
- PBS
- 0.1% Tween
- 4 U/mL RNasin