Hosuk:LabNotes/2014-4-14
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PGP1F sample for 13k Padlock probe test (--> Agi26k0gap)[edit]
- Continued from 04/10
Ampligase[edit]
- Matt’s Agi26k0gap Padlock Probe set
- Padlock probes (16.5pmole) : 97uL (--> 170pM total --> 13 fM per each)
- 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[edit]
- 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[edit]
- 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[edit]
- 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
- Temp. result(04/14) : DecodingResults_v2.xlsx