Matt:LabNotes/2014-2-25: Difference between revisions
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>Mzcai m (→Procedure) |
>Mzcai m (→Procedure) |
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===Procedure=== | ===Procedure=== | ||
====Anneal Padlock Probes==== | |||
*1X Ampligase Buffer | *1X Ampligase Buffer | ||
*2uM of each padlock probe | *2uM of each padlock probe | ||
Line 12: | Line 12: | ||
**Wrapped in parafilm and put in ziploc with wet towel | **Wrapped in parafilm and put in ziploc with wet towel | ||
====Fixed Template - 25U Ampligase (C9 & F9)==== | =====Fixed Template - 25U Ampligase (C9 & F9)===== | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Component''' | | align="center" style="background:#f0f0f0;"|'''Component''' | ||
Line 28: | Line 28: | ||
|} | |} | ||
====Fixed Template - 50U Ampligase (C10 & F10)==== | =====Fixed Template - 50U Ampligase (C10 & F10)===== | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Component''' | | align="center" style="background:#f0f0f0;"|'''Component''' | ||
Line 44: | Line 44: | ||
|} | |} | ||
====1st Rolony (E6 & F6)==== | =====1st Rolony (E6 & F6)===== | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Component''' | | align="center" style="background:#f0f0f0;"|'''Component''' | ||
Line 64: | Line 64: | ||
|} | |} | ||
====Empty Negative Control (D7 & E7)==== | =====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 | *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)==== | =====Pre-circularized Padlock Probes (C7 & C8)===== | ||
*Combined all the pre-circle ppMALAT1 made on: [[Matt:LabNotes/2014-2-10]] into 64ul | *Combined all the pre-circle ppMALAT1 made on: [[Matt:LabNotes/2014-2-10]] into 64ul | ||
*75% EtOH for 10 minutes | *75% EtOH for 10 minutes | ||
Line 75: | Line 75: | ||
*Add 32ul to C7 & C8 | *Add 32ul to C7 & C8 | ||
====Fixed Pre-circularized Padlock Probes (F7 & F8)==== | =====Fixed Pre-circularized Padlock Probes (F7 & F8)===== | ||
*Not going to do anything during Ampligase step | *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(100 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. |
Revision as of 02:12, 27 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
- Pre-annealing the FISSEQ_ppRCA primer for the rolling circle amplification reaction.
- 3ul of RCA primer(100 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.