Hosuk:LabNotes/2013-9-12: Difference between revisions
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====Detail procedure==== | ====Detail procedure==== | ||
*Prepare two barcoded padlock probes - ACTB, RAB7A | *Prepare two barcoded padlock probes - ACTB, RAB7A | ||
=====for detecting ACTB===== | =====for detecting ACTB===== | ||
*ppACTB : /5Phos/CTGTGCTCGCGGGGCG CTTCAGCTTCCCGATATC CGACGG ACGTATCGGTAGTCGCAACGCA GGCAAAGGCGAGGCT --> 77bp | *ppACTB : /5Phos/CTGTGCTCGCGGGGCG CTTCAGCTTCCCGATATC CGACGG ACGTATCGGTAGTCGCAACGCA GGCAAAGGCGAGGCT --> 77bp | ||
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**site for decoding probes (dcProbe0-Cy3) : ACGTATCGGTAGTCGCAACGCA --> 22bp | **site for decoding probes (dcProbe0-Cy3) : ACGTATCGGTAGTCGCAACGCA --> 22bp | ||
**site for 2nd RCA probes : CTTCAGCTTCCCGATATC --> 18bp | **site for 2nd RCA probes : CTTCAGCTTCCCGATATC --> 18bp | ||
=====for detecting RAB7A===== | =====for detecting RAB7A===== | ||
*ppRAB7A: /5Phos/GAAGCGAGAAGGTCCAAGTTCTG CTTCAGCTTCCCGATATC CGACGG GTCTTGCGTGCGATACGGAGTA GAGGAGACTAAACGGAGGACA --> 90bp | *ppRAB7A: /5Phos/GAAGCGAGAAGGTCCAAGTTCTG CTTCAGCTTCCCGATATC CGACGG GTCTTGCGTGCGATACGGAGTA GAGGAGACTAAACGGAGGACA --> 90bp | ||
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* | =====FISSEQ_ppRCA===== | ||
*GATATCGGGAAGCTGA*A*G --> 18bp | |||
*Start at 09/10 | *Start at 09/10 |
Revision as of 17:57, 12 September 2013
Gene decoding with Barcoded Padlock probes
Experimental plan
- Hybridizing padlock probes on 1st rolonies, and
- Generate 2nd rolonies over 1st rolonies, and
- Observe each different fluorescent signal from 2nd rolonies
Detail procedure
- Prepare two barcoded padlock probes - ACTB, RAB7A
for detecting ACTB
- ppACTB : /5Phos/CTGTGCTCGCGGGGCG CTTCAGCTTCCCGATATC CGACGG ACGTATCGGTAGTCGCAACGCA GGCAAAGGCGAGGCT --> 77bp
- two arms : CTGTGCTCGCGGGGCG, GGCAAAGGCGAGGCT
- site for decoding probes (dcProbe0-Cy3) : ACGTATCGGTAGTCGCAACGCA --> 22bp
- site for 2nd RCA probes : CTTCAGCTTCCCGATATC --> 18bp
for detecting RAB7A
- ppRAB7A: /5Phos/GAAGCGAGAAGGTCCAAGTTCTG CTTCAGCTTCCCGATATC CGACGG GTCTTGCGTGCGATACGGAGTA GAGGAGACTAAACGGAGGACA --> 90bp
- two arms : GAAGCGAGAAGGTCCAAGTTCTG, GAGGAGACTAAACGGAGGACA
- site for decoding probes (dcProbe1-Cy3) : GTCTTGCGTGCGATACGGAGTA --> 22bp
- site for 2nd RCA probes : CTTCAGCTTCCCGATATC --> 18bp
FISSEQ_ppRCA
- GATATCGGGAAGCTGA*A*G --> 18bp
- Start at 09/10