Matt:LabNotes/2014-1-30: Difference between revisions
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>Mzcai m (→Results) |
>Mzcai m (→Results) |
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**Majority of signal after blocking is overlapped(red) or is very close to previous signal (green near blue) | **Majority of signal after blocking is overlapped(red) or is very close to previous signal (green near blue) | ||
*Because of the decrease in fluorescent points and most of the ones left overlap with ones before blocking, it shows they aren't random noise and were successfully blocked with an oligonucleotide of similar sequence | *Because of the decrease in fluorescent points and most of the ones left overlap with ones before blocking, it shows they aren't random noise and were successfully blocked with an oligonucleotide of similar sequence | ||
[[File:C10_Blocked_Overlap_C10.png]] | [[File:C10_Blocked_Overlap_C10.png|450px]] |
Latest revision as of 01:08, 5 February 2014
Block Shifted[edit]
- Block Shifted FISSEQ_2ndRCAprimerDye with RT primer oligonucleotide to see if they are hybridizing to the same locations on primary rolonies
Procedure[edit]
- Stripped with 80% formamide for 10min at 50C
- Hybridize RT primer 1uM (2ul 100uM Dye + 60ul 100% formamide + 20ul 20X SSC + 118ul H2O)
- Wash with 2X SSC twice
- Hybridize Shifted FISSEQ 2uM (2ul 200uM Dye + 60ul 100% formamide + 20ul 20X SSC + 118ul H2O)
- Wash with 2X SSC twice
- In the future, the blocking oligonucleotide should be atleast one order of magnitude greater in concentration
Results[edit]
24hrs | Blocked |
289 | 107 |
116 | 68 |
359 | 366 |
21370 | 3213 |
- C10: Blocked(green) overlapping 24hrs (blue)
- Overlap is red
- Majority of signal after blocking is overlapped(red) or is very close to previous signal (green near blue)
- Because of the decrease in fluorescent points and most of the ones left overlap with ones before blocking, it shows they aren't random noise and were successfully blocked with an oligonucleotide of similar sequence