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 70ul
  • 75% EtOH for 10 minutes
  • Wash with 1X PBS 2 times
  • 0.1N HCl for 2 minutes
  • Wash with 1X PBS 3 times
  • Add 35ul to C7 & C8

Fixed Pre-circularized Padlock Probes (F7 & F8)

  • Not going to do anything during Ampligase step