Daniel:Protocols/LatchPadlock: Difference between revisions
Jump to navigation
Jump to search
>Djacobse |
>Djacobse |
||
Line 18: | Line 18: | ||
</ol> | </ol> | ||
<li>Wash twice</li> | <li>Wash twice</li> | ||
<ol type="a"><li>Wash by pelleting cells at 600xg for 3 min</li></ol> | <ol type="a"><li>Wash by pelleting cells at 600xg for 3 min</li></ol></ol> | ||
<li>Circularization</li> | <li>Circularization</li> | ||
<ol type="A"> | <ol type="A"> | ||
<li>Setup up reaction well with:</li> | <li>Setup up reaction well with:</li> | ||
<ol type="a"> | <ol type="a"> | ||
<li>100 | <li>20% v/v HemoKlentaq</li> | ||
<li> | <li>0.5 U/μL Ampligase</li> | ||
<li>100 μM of dNTP mix</li> | |||
<li>1x Ampligase Buffer</li> | |||
</ol> | </ol> | ||
</ol> | </ol> |
Revision as of 22:58, 11 June 2016
Latch and Padlock Hybridization
Protocol
- Probe Hybridization
- Start from hybridized C Probes or antibodies
- Make sure probes have been washed as per the protocol
- After washing, incubate with 100 nM insert/backbone oligos for 30 min at 37C
- Alternative Protocol
- Start with 94C for 30 seconds
- Decrease to 55C at a rate of 0.02C/sec
- Hold at 55C for 20 hours
- Wash twice
- Wash by pelleting cells at 600xg for 3 min
- Circularization
- Setup up reaction well with:
- 20% v/v HemoKlentaq
- 0.5 U/μL Ampligase
- 100 μM of dNTP mix
- 1x Ampligase Buffer
- Insert/backbone Hybridization Buffer
- 100 nM insert/backbone oligos
- 1X SSC
- 40 U/mL RNasin
- PBS
- Wash Buffer
- PBS
- 0.1% Tween
- 4 U/mL RNasin