Matt:LabNotes/2014-1-24: Difference between revisions
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>Mzcai (Created page with "===Generating Primary Rolonies in 32 Wells=== *Hosuk fixed PGP1F cells in 32 wells (rows C-F, columns 3-10) of 96 well plate *I followed standard procedures to generate primar...") |
>Mzcai |
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**Row E: 510bp from TSS | **Row E: 510bp from TSS | ||
**Row F: 1000bp from TSS | **Row F: 1000bp from TSS | ||
====RT RAB7A Results==== | |||
*Previously Hosuk (using FISSEQ_Adpt dye probe)found that primers of different distances from RAB7A TSS produced different numbers of primary rolonies | |||
**Increasing distance from TSS correlated with increasing # of primary rolonies with 1000bp having even more rolonies than produced by random hexamer primer | |||
**However using a dye probe specific for RAB7A target (RAB7A_1stRolonyFISH) which should be on all RAB7A primary rolonies since it is within 100bp of TSS no signal was found | |||
***This is why experiment is worth repeating | |||
Checked here: [[Matt:LabNotes/2014-1-28]] |
Latest revision as of 01:12, 29 January 2014
Generating Primary Rolonies in 32 Wells[edit]
- Hosuk fixed PGP1F cells in 32 wells (rows C-F, columns 3-10) of 96 well plate
- I followed standard procedures to generate primary rolonies
- Used half the concentration of Riboshredder because there wasn't enough left
- Columns 3-8 used random hexamer for RT
- Columns 9-10 used primers that are complementary to RAB7A mRNA a known distance from TSS
- Row C: 220bp from TSS
- Row D: 300bp from TSS
- Row E: 510bp from TSS
- Row F: 1000bp from TSS
RT RAB7A Results[edit]
- Previously Hosuk (using FISSEQ_Adpt dye probe)found that primers of different distances from RAB7A TSS produced different numbers of primary rolonies
- Increasing distance from TSS correlated with increasing # of primary rolonies with 1000bp having even more rolonies than produced by random hexamer primer
- However using a dye probe specific for RAB7A target (RAB7A_1stRolonyFISH) which should be on all RAB7A primary rolonies since it is within 100bp of TSS no signal was found
- This is why experiment is worth repeating
Checked here: Matt:LabNotes/2014-1-28