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====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)===== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X Ampligase Buffer||10 |- | 200uM ppMALAT1||1 |- | 5U/ul Ampligase||10 |- | H2O||79 |- | Total||100 |} =====Fixed Template - 50U Ampligase (C10 & F10)===== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X Ampligase Buffer||10 |- | 200uM ppMALAT1||1 |- | 5U/ul Ampligase||20 |- | H2O||69 |- | Total||100 |} =====1st Rolony (E6 & F6)===== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''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
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