Hosuk:LabNotes/2013-9-26
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ACTB Gene detection in 1st Rolonies[edit]
Procedure[edit]
- Directly anneal ATTO488_ACTB_on1stRolony_target_Probe.
- Use 06232013 sample – 3uL Phi29 (2nd rolony experiment sample) and 10uL Phi29
- Procedure
- Strip Sample dishes with 80% Formamide
- Prepare 1uM Alex288_ACTB_on1stRolony_target_Probe in 200uL 2x SSC
- Preheat the probe at 65C for 5min
- Add probes in dish, incubate for 10min at RT
- Wash, and image
- ACTB_1stRolonyFISH : GGCAAAGGCGAGGCT/3ATTO488N/
- Shifted FISSEQ_2ndRCAprimerDye : GGATATCGGGAAGCTGAA/3ATTO488N/
Result[edit]
- Too few, ACTB itself might not be many on entire RNAs.
- Matt has found a data (microarray), and 5% out of RNA in PGP1F is mRNA, and 1% of mRNA is ACTB. According to this, there are very few of ACTB in 1st rolonies.
Next Plan[edit]
- Find other gene which would be plenty in PGP1F
- Or try RAB7A
Pictures[edit]
1st Rolonies, 06/23, 11th Try, 3uL Phi29 | 1st Rolonies, 06/23, 11th Try, 10uL Phi29 | ||
File:S3 3uLPhi29Enzymat 20x-NA0.8 pos01 Cy3 exp0.1-EM00.jpg | File:S4 10uLPhi29Enzymat 20x-NA0.8 pos03 Cy3 exp0.1-EM5.jpg | ||
ACTB on 1st Rolonies, 3uL Phi29 sample (raw) | ACTB on 1st Rolonies, 10uL Phi29 sample (raw) | ||
File:ACTB 1stRolonyTarget FITC 0623-3uLPhi29 pos01 fig01 FITC.jpg | File:ACTB 1stRolonyTarget FITC 0623-10uLPhi29 pos02 fig01 FITC.jpg |