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=Bead with PKP2 Target for DARTFISH Positive Control= *Using PA gel may prevent diffusion of probes and enzymes during DARTFISH *To test this and to have a positive control for future experiments need to design a synthetic target **Attach target to magnetic streptavidin bead (Dynabeads MyOne C1) *Design oligonucleotide with biotin 5' modification **Chose PKP2 gene because haven't detected it in any DARTFISH samples **Also this specific padlock probe has very high efficiency when measured in tube PKP2_control /5BiosG/AAAAAAGAGATGGCTGTCTTTTTCACACTTGGGTCACCAACATGCAGCATCTTTC ==Link PKP2_control oligo to Streptavidin Bead== #Make 2X B&W (Binding and Wash) Buffer #*16ml 5uM NaCl + 80ul 5uM EDTA + 100ul 4uM Tris-HCl + 23.82ml H2O #Resuspend beads by vortexing #Transfer 100ul of beads (10ug/ul) to new tube #Pull down by magnet 2min and remove supernatant #Wash 3x with 100ul 1X B&W Buffer by resuspending and then removing supernatant #Resuspend in 200ul 2X B&W Buffer #Add 200ul of 2.5uM PKP2_Control #Incubate 15min at RT gently rotating #Pull down with magnet #Wash 3x with 1X B&W Buffer #Resuspend in 1ml 1X PBS ==Measure DNA Concentration== *Use Qubit ssDNA kit to measure amount of ssDNA on beads **Assume free biotin-oligos have been all washed away **Measure only streptavidin bead as well for base-line *Beads + DNA: 1.56ng/ul *Beads only: 164pg/ul *Don't trust the quantitation but it shows ssDNA was bound to beads ==Capture with CA12kNov2014 V4 in tube== [[Matt:LabNotes/2015-5-18 | Reference protocol]] *Use CA12kNov2014_V4 Probes to Capture PKP2_Control oligos attached to streptavidin beads to test ====Sample Groups==== #PKP2_Control on beads #Beads only (NTC) {| {{table}} | align="center" style="background:#f0f0f0;"|'''Sample #''' | align="center" style="background:#f0f0f0;"|'''Sample Description''' | align="center" style="background:#f0f0f0;"|'''Probes (ul)''' | align="center" style="background:#f0f0f0;"|'''Target (ul)''' | align="center" style="background:#f0f0f0;"|'''Target Conc (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''10X Ampligase Buffer''' | align="center" style="background:#f0f0f0;"|'''H2O''' | align="center" style="background:#f0f0f0;"|'''Total''' |- | 1||PKP2_Control||3.5||1||1.56||3||22.5||30 |- | 2||NTC||3.5||1||0||3||22.5||30 |} *Add 40ul Mineral Oil on top '''Program'''<br> * 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 20h **In BioRad Thermalcycler program says: -0.2C per cycle every 30sec * -> add 3ul AmpLigase enzyme mix (0.5U/ul AmpLigase in 1X AmpLigase buffer) * -> 55 C 20h (actually 24h)-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold. ====AmpLigase enzyme mix==== {| class="wikitable" style="text-align:center;{{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Stock conc.''' | align="center" style="background:#f0f0f0;"|'''Unit''' | align="center" style="background:#f0f0f0;"|'''Final conc.''' | align="center" style="background:#f0f0f0;"|'''Unit''' | align="center" style="background:#f0f0f0;"|'''Prepare volume 30ul''' |- | AmpLigase||5||U/ul||0.5||U/ul||2.00 |- | 10x AmpLigase Buffer||10||x||1||x||2.00 |- | H2O||||||||||16.00 |- | Total||||||||||20.00 |} ==PCR + Sequencing Adapters== ====Primers==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Primer''' | align="center" style="background:#f0f0f0;"|'''Sequence''' | align="center" style="background:#f0f0f0;"|'''Index #''' |- | ISB_CA_AF||AATGATACGGCGACCACCGAGATCTACACGCCTGCATATCGGGAAGCTGAAG|| |- | ISB_CA_AR.T1||CAAGCAGAAGACGGCATACGAGATCGTGATCGGTCTGCCTTCCCGATATCCGACGG||Indx1 |- | ISB_CA_AR.T2||CAAGCAGAAGACGGCATACGAGATACATCGCGGTCTGCCTTCCCGATATCCGACGG||Indx2 |- | ISB_CA_AR.T3||CAAGCAGAAGACGGCATACGAGATGCCTAACGGTCTGCCTTCCCGATATCCGACGG||Indx3 |} {| {{table}} | align="center" style="background:#f0f0f0;"|'''Sample''' | align="center" style="background:#f0f0f0;"|'''Index''' | align="center" style="background:#f0f0f0;"|'''Forward Primer''' | align="center" style="background:#f0f0f0;"|'''Reverse Primer''' |- | PKPK2_Control||3||ISB_CA_AF||ISB_CA_AR.T3 |- | NTC||1||ISB_CA_AF||ISB_CA_AR.T1 |} ====PCR Test==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1X Volume''' |- | Captured template||10 |- | 10uM Forward Primer||0.4 |- | 10uM Reverse Primer||0.4 |- | 2X KAPA SYBG MM||12.5 |- | H2O||1.7 |- | Total||25 |} *Used 10ul of template instead of 1ul because expect low amount due to only target for 1 of the 3,500 padlock probes Program 98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x25 [[File:2016-04-04_PadlockProbe_PKP2_in_tube_Test_Sequence_Adapter_PCR.JPG| 450px]] ====PCR==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1X Volume''' |- | Captured template||12 |- | 10uM Forward Primer||2 |- | 10uM Reverse Primer||2 |- | 2X KAPA SYBG MM||50 |- | H2O||34 |- | Total||100 |} *Split into duplicates of 50ul because Biorad cycler can't handle >50ul reaction volume Program 98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min [[File:2016-04-05_PadlockProbe_PKP2_in_tube_Sequence_Adapter_PCR.JPG|450px]] *Bead purification with 1.4:1 Beads to amplicon volume ratio **Eluted with 50ul total for each sample ===TBU Gel Check=== *Load 2ul of each sample + 2ul loading dye [[File:20160406_PP_capture_seq_library.jpg|150px]] *Lane 1: Low Mass Ladder *Lane 2: Erin's capture of gDNA from CA12kNov2014_V4 that had 3 extra cycles during Production PCR *Lane 3: Erin's capture of NTC from CA12kNov2014_V4 that had 3 extra cycles during Production PCR *Lane 4: PKP2_Control captured ===Gel Size Selection=== ===TBU Gel Check===
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