Hosuk:LabNotes/2013-9-12: Difference between revisions
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==== | ====Procedure==== | ||
*Prepare two barcoded padlock probes - ACTB, RAB7A | *Prepare two barcoded padlock probes - ACTB, RAB7A | ||
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=====2nd RCA probe===== | =====2nd RCA probe===== | ||
*'''FISSEQ_ppRCA''' : GATATCGGGAAGCTGA*A*G --> 18bp | *'''FISSEQ_ppRCA''' : GATATCGGGAAGCTGA*A*G --> 18bp | ||
====Overall Process==== | |||
#Prepare each probes – the concentration of each probes are resuspended to 200uM for each ACTB, RAB7A and 2nd RCA primer. | |||
#Hybridize Padlock probe – mixture: 1uL ACTB + 1uL RAB7A + 98uL 2x SSC | |||
#Add mix, and incubate dish in the oven at 55C for 1hr. | |||
#Prepare Ampligase mix - 1uL AmpLigase + 10uL Buffer + 89uL H2O | |||
#Aspirate PBS in rolony sample and wash with PBS once | |||
#Add AmpLigase mix to sample dish, and incubate in the oven at 45C for 4hr. | |||
#Run 2nd RCA from Padlock probes | |||
*# Pre-anneal RCA primer (2uL of 100uM in 98uL 2x SSC) 60°C for 15min | |||
*# Add RCA master mix with aa-dUTP, run RCA for 20 hr. | |||
# Fix 2nd RCA with BS(PEG)9, and deactivate BS(PEG)9 with Tris pH8.0 treatment | |||
# Check image with Cy3 (for the ACTB or RAB7A gene on 2nd rolonies) and Cy5 channel (1st rolonies) | |||
*Start at 09/10 | *Start at 09/10 |
Revision as of 18:03, 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
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
Overall Process
- Prepare each probes – the concentration of each probes are resuspended to 200uM for each ACTB, RAB7A and 2nd RCA primer.
- Hybridize Padlock probe – mixture: 1uL ACTB + 1uL RAB7A + 98uL 2x SSC
- Add mix, and incubate dish in the oven at 55C for 1hr.
- Prepare Ampligase mix - 1uL AmpLigase + 10uL Buffer + 89uL H2O
- Aspirate PBS in rolony sample and wash with PBS once
- Add AmpLigase mix to sample dish, and incubate in the oven at 45C for 4hr.
- Run 2nd RCA from Padlock probes
- Pre-anneal RCA primer (2uL of 100uM in 98uL 2x SSC) 60°C for 15min
- Add RCA master mix with aa-dUTP, run RCA for 20 hr.
- Fix 2nd RCA with BS(PEG)9, and deactivate BS(PEG)9 with Tris pH8.0 treatment
- Check image with Cy3 (for the ACTB or RAB7A gene on 2nd rolonies) and Cy5 channel (1st rolonies)
- Start at 09/10