Hosuk:LabNotes/2013-9-12: Difference between revisions
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*Prepare two barcoded padlock probes - ACTB, RAB7A | *Prepare two barcoded padlock probes - ACTB, RAB7A | ||
=====for | =====Padlock probe for ACTB===== | ||
*ppACTB : /5Phos/CTGTGCTCGCGGGGCG CTTCAGCTTCCCGATATC CGACGG ACGTATCGGTAGTCGCAACGCA GGCAAAGGCGAGGCT --> 77bp | *'''ppACTB''' : /5Phos/CTGTGCTCGCGGGGCG CTTCAGCTTCCCGATATC CGACGG ACGTATCGGTAGTCGCAACGCA GGCAAAGGCGAGGCT --> 77bp | ||
**two arms : CTGTGCTCGCGGGGCG, GGCAAAGGCGAGGCT | **two arms : CTGTGCTCGCGGGGCG, GGCAAAGGCGAGGCT | ||
**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 | =====Padlock probe for RAB7A===== | ||
*ppRAB7A: /5Phos/GAAGCGAGAAGGTCCAAGTTCTG CTTCAGCTTCCCGATATC CGACGG GTCTTGCGTGCGATACGGAGTA GAGGAGACTAAACGGAGGACA --> 90bp | *'''ppRAB7A''' : /5Phos/GAAGCGAGAAGGTCCAAGTTCTG CTTCAGCTTCCCGATATC CGACGG GTCTTGCGTGCGATACGGAGTA GAGGAGACTAAACGGAGGACA --> 90bp | ||
**two arms : GAAGCGAGAAGGTCCAAGTTCTG, GAGGAGACTAAACGGAGGACA | **two arms : GAAGCGAGAAGGTCCAAGTTCTG, GAGGAGACTAAACGGAGGACA | ||
**site for decoding probes (dcProbe1-Cy3) : GTCTTGCGTGCGATACGGAGTA --> 22bp | **site for decoding probes (dcProbe1-Cy3) : GTCTTGCGTGCGATACGGAGTA --> 22bp | ||
**site for 2nd RCA probes : CTTCAGCTTCCCGATATC --> 18bp | **site for 2nd RCA probes : CTTCAGCTTCCCGATATC --> 18bp | ||
===== | =====2nd RCA probe===== | ||
*GATATCGGGAAGCTGA*A*G --> 18bp | *'''FISSEQ_ppRCA''' : GATATCGGGAAGCTGA*A*G --> 18bp | ||
*Start at 09/10 | *Start at 09/10 |
Revision as of 17:58, 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
Padlock probe for 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
Padlock probe for 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
2nd RCA probe
- FISSEQ_ppRCA : GATATCGGGAAGCTGA*A*G --> 18bp
- Start at 09/10