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====Slide PCR==== *Make PCR Reaction Mix on ice {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' | align="center" style="background:#f0f0f0;"|'''2X Volume''' |- | 100uM SeqRolony_CL_Amplify||1||2 |- | 2X Taq Master Mix||50||100 |- | H2O||49||98 |- | Total||100||200 |} *Place cover-slip on clean glass slide (tissue face up) *Use silicon isolator with circular opening to seal tissue **NegCtrl silicon had large gaps so used glue to glue all around the circle on bottom of silicon [[File:033115_SeqRolony_NegCtrl.JPG|450px]] **Exp had very small leak so used glue to seal the edges of silicon [[File:033115_SeqRolony_ExpBefore.JPG|450px]] *Add 100ul PCR mix and seal with plastic to prevent evaporation [[File:033115_SeqRolony_ExpAfter.JPG|450px]] *Put on thermocycler with slide adapter '''Program''' 90C 3min -> (90C 20sec -> 58C 30sec -> 68C 45sec) x 20 -> 68C 2min -> 15C hold *Remove plastic seal and pipette solution into PCR tubes [[File:033115_SeqRolony_ExpExtract.JPG|450px]] **NegCtrl had ~90ul (good) but Exp was completely dry (bad, probably leaked out as evidenced by white haze on glass slide outside of silicon) *Add 100ul H2O, let sit for 5min, and pipette into empty PCR tubes (to get more amplicon) *'''Final amount:''' 190ul NegCtrl and 100ul Exp
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