Matt:LabNotes/2014-2-25: Difference between revisions
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**Strip with 80% formamide @37C | **Strip with 80% formamide @37C | ||
*To E6 & F6 add 50ul 1uM dcProbe1-Cy3 (10ul 10uM dcProbe1-Cy3 + 30ul 100% formamide + 10ul 20X SSC + 50ul H2O preheated to 75C) | *To E6 & F6 add 50ul 1uM dcProbe1-Cy3 (10ul 10uM dcProbe1-Cy3 + 30ul 100% formamide + 10ul 20X SSC + 50ul H2O preheated to 75C) | ||
*'''Images saved under 2-27-2014 folder''' |
Revision as of 03:44, 28 February 2014
Secondary Rolony Generation
Samples: Matt:LabNotes/2014-2-21
Procedure
Anneal Padlock Probes
- 1X Ampligase Buffer
- 2uM of each padlock probe
- 0.5 U/ul Ampligase
- Incubate @ 45C for 24 hours
- Wrapped in parafilm and put in ziploc with wet towel
Fixed Template - 25U Ampligase (C9 & F9)
Component | Volume |
10X Ampligase Buffer | 10 |
200uM ppMALAT1 | 1 |
5U/ul Ampligase | 10 |
H2O | 79 |
Total | 100 |
Fixed Template - 50U Ampligase (C10 & F10)
Component | Volume |
10X Ampligase Buffer | 10 |
200uM ppMALAT1 | 1 |
5U/ul Ampligase | 20 |
H2O | 69 |
Total | 100 |
1st Rolony (E6 & F6)
Component | Volume |
10X Ampligase Buffer | 10 |
200uM ppMALAT1 | 1 |
200uM ppACTB | 1 |
200uM ppRAB7A | 1 |
5U/ul Ampligase | 10 |
H2O | 77 |
Total | 100 |
Empty Negative Control (D7 & E7)
- Not going to waste Ampligase since it's highly unlikely anything will be ligated, but will do RCA in these wells
Pre-circularized Padlock Probes (C7 & C8)
- Combined all the pre-circle ppMALAT1 made on: Matt:LabNotes/2014-2-10 into 64ul
- 75% EtOH for 10 minutes
- Wash with 1X PBS 2 times
- 0.1N HCl for 2 minutes
- Wash with 1X PBS 3 times
- Add 32ul to C7 & C8
Fixed Pre-circularized Padlock Probes (F7 & F8)
- Not going to do anything during Ampligase step
Secondary RCA
- Aspirate each well but don't wash
- F7 well was dried up
- Pre-annealing the FISSEQ_ppRCA primer for the rolling circle amplification reaction.
- 3ul of RCA primer(200 uM, GATATCGGGAAGCTGA*A*G) in 597ul of 2xSSC/30% formamide
- Preheat primer at 60C (Tm = 55C when not in 30% formamide)
- Add 50ul to each well, incubate at 45C for 15min.
- Aspirate, and wash using 2x SSC twice
- Aspirate, and wash using 0.1x SSC twice
- Prepare the rolling circle amplification reaction mixtures on ice.
- 513 ul ddH2O,
- 60 ul 10x Phi29 buffer,
- 6 ul 25 mM dNTP,
- 12 ul 2 mM aminoallyl dUTP
- 9 ul Phi29 DNA polymerase (Epicentre, low concentration)
- 600ul Total Volume
- Add 50ul to each well and incubate overnight at 30C. (Put in at 7pm 2/26/14)
- Wash 1X PBS once
- Add 50ul BS(PEG)9 mix to each well and incubate @RT for 1hr (12ul BS(PEG)9 + 588ul 1X PBS)
- Wash 1X PBS twice
- Add 50ul 1M Tris pH 8.0 @RT for 1hr
- Wash 1X PBS twice
Detection
- To all wells add 50ul 1uM dcProbe2-Cy3 (6ul 100uM dcProbe2-Cy3 + 180ul 100% formamide + 60ul 20X SSC + 354ul H2O preheated to 75C)
- Strip E6 & F6 with 80% formamide @ 37C
- To E6 & F6 add 50ul 1uM dcProbe0-Cy3 (10ul 10uM dcProbe0-Cy3 + 30ul 100% formamide + 10ul 20X SSC + 50ul H2O preheated to 75C)
- Strip with 80% formamide @37C
- To E6 & F6 add 50ul 1uM dcProbe1-Cy3 (10ul 10uM dcProbe1-Cy3 + 30ul 100% formamide + 10ul 20X SSC + 50ul H2O preheated to 75C)
- Images saved under 2-27-2014 folder