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==in vitro Capture with CA12k_Nov2014 + suppressor oligo v2 Probe Set== *[[Matt:LabNotes/2015-4-28|Last time captured HBRR cDNA]] with 39 suppressors [[Matt:LabNotes/2015-5-8#Comparison|was not effective for most highly captured genes]] so [[Matt:LabNotes/2015-5-8#Design_Additional_Suppressor_Oligos|made 5 more with new design]] that is hopefully more effective *Will capture [[Matt:LabNotes/2015-5-15|cDNA]] from BA8 tissue **Using RNA from Blue's bulk RNA-Seq experiment **2 versions of cDNA: ***RT with dT (poly dT) ***RT with N9 (random nonamer) ===Sample Groups=== #gDNA 12878 (80.3ng/ul) #*Also a positive control and will be done using same volumes/amounts as all previous experiments #cDNA dT BA8 #*~1/10th of gDNA amount #*cDNA N9 BA8 #*~1/10th of gDNA amount #Negative Control #*From [[Matt:LabNotes/2015-5-15|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||3514||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 (3514, 150nt)||1.71585106x10^8||g/mol |- | Amount Probe req'd||26.4||ng |} *[[Matt:LabNotes/2015-5-11#Qubit_Quantification|461nM]] x (150nt*325Da/nt + 79Da) x 10^-6 L/ul = 22.5 ng/ul **Dilute to 16ul of 2.25ng/ul **10X less probes in cDNA than gDNA *Make suppressor oligo pools with 5nM and 50nM of each supp oligos **1000-fold more of each suppressor oligo: 1.5385x10^-16 mole * 1000 = 1.5385x10^-13 mole = 1.5385x10^-4 nmole = 50 nM * 3.1ul **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||12||3.8||3.1 (50nM each)||3||8||30 |- | 2||cDNA dT||1.2||20||3.1 (5nM each)||3||2.7||30 |- | 3||cDNA N9||1.2||20||3.1 (5nM each)||3||2.7||30 |- | 4||NTC||1.2||20||3.1 (5nM each)||3||2.7||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''' |- | 1||1||ISB_CA_AF||ISB_CA_AR.T1 |- | 2||2||ISB_CA_AF||ISB_CA_AR.T2 |- | 3||3||ISB_CA_AF||ISB_CA_AR.T3 |- | 4||1||ISB_CA_AF||ISB_CA_AR.T1 |} ====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 |} *Aliquot 23.6ul from 4.5X master mix and add 1ul captured template and 0.4ul corresponding reverse primer Program 98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x21 [[File:20150521_CA12kNov14suppv2_invitroPCRtest.JPG| 650px]] *cDNA captured samples look barely better than NTC ====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 |} *Aliquot 86ul from 4.5X master mix and add 12ul captured template and 2ul corresponding reverse primer Program 98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x27 -> 72C 3min *Take gDNA out after 21 cycles *Take cDNA_dT out after 31 cycles *Take cDNA_N9 out after 27 *NegCtrl had 34 cycles [[File:20150521_CA12kNov14suppv2_invitroPCR.JPG|650px]] *During PCR the curves had not decreased (only slightly plateau'd) when I took them out *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:2015-05-22_CA12kNov2014suppv2_invitro_gelcheck.jpg|450px]] *Labeled tubes and put in "Non-quantified Sequencing Libraries" **Sample1: MC20150522_CA12kNov14suppv2_gDNA-1 **Sample2: MC20150522_CA12kNov14suppv2_cDNAdT-2 **Sample3: MC20150522_CA12kNov14suppv2_cDNARan-3 **Sample4: MC20150522_CA12kNov14suppv2_NegCtrl-1 ==Analysis== *Continued here: [[Matt:LabNotes/2015-7-12]]
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