Matt:LabNotes/2014-2-25
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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
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