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==in vitro Capture with TB12k_Apr2016 V4== *Will capture [[Matt:LabNotes/2016-6-4|cDNA]] from BA8 tissue **Using RNA from Blue's bulk RNA-Seq experiment ===Sample Groups=== #gDNA 12878 (80.3ng/ul) #*Also a positive control and will be done using same volumes/amounts as all previous experiments #*cDNA N9 BA8 #*~1/10th of gDNA amount #Negative Control #*From [[Matt:LabNotes/2015-6-4|cDNA synthesis procedure]] where no RTase was added #*If see capture product, then there was DNA in Blue's RNA sample ====gDNA==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Probe:target''' | align="center" style="background:#f0f0f0;"|'''1000:1''' | align="center" style="background:#f0f0f0;"|'''''' |- | Probe size||4978||probes |- | DNA templet||300||ng |- | gDNA MW||1.95x10^12||g/mol |- | gDNA (300ng) ||1.5385x10^-19||mol |- | Probe (1000:1)||1.5385x10^-16||mol |- | Probe MW (4978, 157nt)||2.37748782x10^8||g/mol |- | Amount Probe req'd||36.6||ng |} *[[Matt:LabNotes/2016-5-25#Qubit_Quantification|423nM]] **Dilute 22ul to 3.66ng/ul (2.2ul probe + 19.8ul H2O) **10X less probes in cDNA than gDNA {| {{table}} | align="center" style="background:#f0f0f0;"|'''Sample #''' | align="center" style="background:#f0f0f0;"|'''Sample Description''' | align="center" style="background:#f0f0f0;"|'''Probes''' | align="center" style="background:#f0f0f0;"|'''Target''' | align="center" style="background:#f0f0f0;"|'''1000X Supp Oligos''' | align="center" style="background:#f0f0f0;"|'''10X Ampligase Buffer''' | align="center" style="background:#f0f0f0;"|'''H2O''' | align="center" style="background:#f0f0f0;"|'''Total''' |- | 1||gDNA||18||3.8||3||5.2||30 |- | 2||cDNA||1.8||20||3||5.2||30 |- | 3||NTC||1.8||20||3||5.2||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 |} ===Add Sequence Adapters PCR=== ====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''' |- | gDNA||1||ISB_CA_AF||ISB_CA_AR.T1 |- | cDNA||2||ISB_CA_AF||ISB_CA_AR.T2 |- | NTC||3||ISB_CA_AF||ISB_CA_AR.T3 |} ====PCR Test==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1X Volume''' | align="center" style="background:#f0f0f0;"|'''4.5X Volume''' |- | Captured template||1||0 |- | 10uM Forward Primer||0.4||1.8 |- | 10uM Reverse Primer||0.4||0 |- | 2X KAPA SYBG MM||12.5||56.25 |- | H2O||10.7||48.15 |- | Total||25||106.2 |} Program 98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x21 [[File:TB12k_Apr2016_V4_Capture_PCR_test.JPG|450px]] ====PCR==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1X Volume''' | align="center" style="background:#f0f0f0;"|'''4.5X Volume''' |- | Captured template||12||0 |- | 10uM Forward Primer||2||9 |- | 10uM Reverse Primer||2||0 |- | 2X KAPA SYBG MM||50||225 |- | H2O||34||153 |- | Total||100||387 |} Program 98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x26 -> 72C 3min -> 4C hold *Take gDNA out after 25 cycles *Take cDNA out after 30 cycles [[File:TB12k_Apr2016_V4_Capture_PCR.JPG|450px]] *Bead purification with 1.5:1 Beads to amplicon volume ratio **Eluted with 50ul total for each sample ===PAGE Check=== *Load 2ul of each sample + 2ul loading dye [[File:2016-06-13_TB12kApr2016_V4_intubecapture_gelcheck.jpg|250px]] *Bead purification did not work try again with 0.8:1 bead:sample ratio [[File:2016-06-13_TB12kApr2016_V4_intubecapture_gelcheckRnd2.jpg|250px]] *Labeled tubes and put in "Non-quantified Sequencing Libraries" **Sample1: MC20160610_TB12kApr16_gDNA-1 (MC160610_TB12kApr_gDNA-1) **Sample2: MC20150610_TB12kApr16_cDNA-2 (MC160610_TB12kApr_cDNA-2)
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