Hosuk:LabNotes/2014-4-14: Difference between revisions
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>Hosuki78 (Created page with "*LabNote ===PGP1F sample for 13k Padlock probe test=== *Continued from 04/10 ====Ampligase==== *Matt’s 13k, 0 gap Padlock ...") |
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*[[Hosuk:Lab_Notes|LabNote]] | *[[Hosuk:Lab_Notes|LabNote]] | ||
===PGP1F sample for 13k Padlock probe test=== | ===PGP1F sample for 13k Padlock probe test (--> Agi26k0gap)=== | ||
*Continued from [[Hosuk:LabNotes/2014-4-10|04/10]] | *Continued from [[Hosuk:LabNotes/2014-4-10|04/10]] | ||
====Ampligase==== | ====Ampligase==== | ||
*Matt’s | *Matt’s Agi26k0gap Padlock Probe set | ||
**Padlock probes (16.5pmole) : 97uL | **Padlock probes (16.5pmole) : 97uL | ||
**Ampligase Buffer : 15uL | **Ampligase Buffer : 15uL |
Revision as of 20:08, 15 April 2014
PGP1F sample for 13k Padlock probe test (--> Agi26k0gap)
- Continued from 04/10
Ampligase
- Matt’s Agi26k0gap Padlock Probe set
- Padlock probes (16.5pmole) : 97uL
- Ampligase Buffer : 15uL
- Ampligase Enzyme : 15uL
- H2O : 23uL
- Total 150 uL
- Start Ampligase: 04/11/14, 10:30am, incubate for 48 hr.
2nd RCA reaction
- RCA Mix
- H2O : 162uL
- 10x Buffer : 20uL
- dNTP : 4uL
- adUTP : 8u:
- Phi29 : 6uL
- Start RCA : 04/13/14 ~ 04/14/14 for 24hr.
Imaging & Decoding
- Imaging : 04/14/14
- 1uM of FAM, and Cy3 in 100uL total dcProbes volume
- 10uL of FAM + 10uL of Cy3 + 80uL of 2x SSC
- 20x obj. 2048 x 2048 resolution
- Laser : 5% for both 488, 532nm
- PMT gain : 610
- Step0 : Cy3-1st Rololy detection
- Step1 : dcProbe0-FAM, dcProbe0-Cy3
- Step1 : dcProbe1-FAM, dcProbe1-Cy3
...
- Step5 : dcProbe4-FAM, dcProbe4-Cy3
- Strip with 80% formamide at inbetween each step (for 15min incubation)
Decoding Result
- Each image at each step showed reasonable number of signals, but not many.
- 1st Rolony signal was too much decreased compared to the first observation at 04/10... WHY?
- Analysis is on going