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==Sequence in situ Rolonies via in situ Slide PCR== *Purpose: Sequence the rolonies in situ to quantify the contributions of each probe to the decoded rolonies **ie gene GNG4 has many rolonies but are they due to a single highly efficient probe or a number of different probes *Primer design and general method: [[Matt:LabNotes/2015-3-14]] ===Protocol=== *Samples: **Exp: BA8 CA12k_Nov2014_V4 Captured Rolonies **NegCtrl: BA8 1st (cDNA) Rolonies (from old FISSEQ protocol) ====Strip hybridized dye-probes with 80% formamide pre-heated to 80C==== *The tissue is on thin cover-slip placed on a glass slide *Take cover-slip off glass slide and put in small culture dish *Incubate for 15min at RT and then wash twice with 1X PBS [[File:033115_SeqRolony_Strip.JPG|450px]] ====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 ====Add Sequencing Adapters==== *Qiagen PCR purification **Elute 40ul *Nanodrop ssDNA: **Exp: 0.6 ng/ul **NegCtrl: 2.2 ng/ul ====PCR Test==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1X Volume''' | align="center" style="background:#f0f0f0;"|'''3X Volume''' |- | Slide PCR amplicon||11.7||0 |- | SeqRolony_P5||0.4||1.2 |- | SeqRolony_P7||0.4||1.2 |- | 2X KAPA SYBG MM||12.5||37.5 |- | Total||25||60 |} *Aliquot 13.3ul from 3X master mix and add 11.7ul slide amplicon Program 98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min [[File:033115_SeqRolony_PCRTest.JPG]] ====Gel Check==== *TBE Gel with 4ul sample + 4ul 6X Loading Dye **0.5ul Low Mass Ladder [[File:2015-04-01_SeqRolony_PCRtest.jpg|150px]] *Main bright band is the correct size (~225bp <- 150bp padlock probe + 75bp adapters) ====PCR==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1X Volume''' | align="center" style="background:#f0f0f0;"|'''2.5X MM''' |- | Slide PCR amplicon||27||0 |- | SeqRolony_P5||1||2.5 |- | SeqRolony_P7||1||2.5 |- | 2X KAPA SYBG MM||50||125 |- | H2O||21||52.5 |- | Total||100||187.5 |} *Aliquot 73ul from 2.5X master mix and add 27ul captured template Program 98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x9 -> 72C 3min [[File:040115_SeqRolony_PCR.JPG|650px]] *Qiagen PCR Purification **Elute 40ul *Nanodrop measure concentration **Exp: 45.5 ng/ul * 38ul = 1.729ug **NegCtrl: 27.9 ng/ul * 38ul = 1.060ug ====Size Selection==== *5-well TBE Gel 220V for 32min **Low mass ladder **3 lanes of Exp **1 lane of NegCtrl *Cut out the bright band at 225bp [[File:2015-04-02_SeqRolony_SizeSelect.jpg|650px]] *Add to 2 0.5mls tube (with hole at bottom) in 2ml tubes **Spin at 15,000rpm for 3 min *Add 450ul 1X TE Buffer to each and shake at 37C for 1 hour *Filter out gel with 2 Nanosep columns **Spin at 15,000rpm for 3 min **Transfer solution, spin, then transfer gel and spin *Add 1000ul 100% EtOH, 45ul 3M NaOAc, and 1.4ul glycoblue **Vortex *Put in -80C freezer for 30min *Spun at 10,000rpm at 4C for 30min *Discard supernatant and add 650ul cold 80% EtOH and spin at 14,000rpm at 4C for 5min *Discard supernatant and let dry in hood for 10min *Resuspend each tube with 15ul and combine *'''FINAL:''' 30ul in 1.5ml tube labeled "MC-SeqRolony_BA8V4_Mar3115-13" and put in Non-quantified New Sequencing Libraries box with SeqRolony_RP1 and SeqRolony_IRP
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