Revision as of 02:26, 27 February 2014 by >Mzcai
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
- 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)
- Add 50ul to each well and incubate overnight at 30C.