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===Test Decoding probes using custom pre-circularized oligos=== ====Previous try==== *Alan and I have tried decoding pre-circularized oligos made from Matt at 07/30/2013. *I added oligos in fixed cells on MatTek glass-bottom dish, and ran RCA to generate rolonies. *However the signal wasn’t seen any FL signal after hybridizing decoding probes. *We suspected that the amount of oligos might not enough (The actual concentration or amount of the oligos was unknown). *So I've ordered 90bp ssDNA from IDT which has 3 sites for decoding probes and 1 site for RCA probes. ====2nd try with 90bp custom oligo==== =====Template design===== *Sequence : /5Phos/CCCGATATCCGACGGTAGTGTGTCTTGCGTGCGATACGGAGTATCTACTTCGTCGCGTCAGACCATCGGAATACGTCGTTGACTGCGTTC *RCA probe sequence : ACACTACCGTCGGATATC *Decoding probes for this experiment **dcProbe1-Cy3 : Cy3-GTCTTGCGTGCGATACGGAGTA **dcProbe2-Cy3 : Cy3-TCTACTTCGTCGCGTCAGACCA **dcProbe4-FAM : FAM-TCGGAATACGTCGTTGACTGCG [[File:Sequence_Template_Probes_.png|500px]] =====Circularization===== *CircLigase II reaction with 50pmole input DNA **6 hr reaction [[File:CircLigaseII_20uL.png|300px]] *Circ. reaction lane has no band near 90bp, it seemed circularized. [[File:CircLigaseII_20uL_Gel.png|250px]] =====Rolony generation===== *Add template, incubate 15min *Run RNase H +Riboshredder for 1hr at 60C. *Run RCA for 20hr at 30C =====Run Cycle with 3 probes===== *Procedure : Hybridize Probe1-Cy3 --> Image --> Strip --> Image --> Probe4-FAM --> Image --> Strip --> Image --> Probe2-Cy3 --> Image --> … *Cy3 signal looked good, but FAM signal was weak, and fewer numbers. *Second Cy3 probes were fewer than first one, it was because of less non-specific binding due to Formamide…?? *We'll try using confocal for FAM signal imaging, and try Matt's oligo one more time. [[File:Probes_CycleRun12.png|800px]] [[File:Probes_CycleRun34.png|800px]] [[File:Probes_CycleRun567.png|800px]] *'''Overlay''' [[Media:Composite_Probe1-Cy3_Probe2-Cy3.jpg|Probe1-Cy3 and Probe2-Cy3]], [[Media:Composite_Probe2-Cy3_Probe4-FAM.jpg|Probe2-Cy3 and Probe4-FAM]] [[File:Probes_CycleRun_Composites.png|800px]]
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