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==in vitro Capture with CA12k_Nov2014 Probe Set + Supp Oligos== ===HBRR cDNA Synthesis=== * Followed NEB E6300S protocol: [https://www.neb.com/protocols/1/01/01/first-strand-cdna-synthesis-e6300] * Human Brain Reference RNA: [https://www.lifetechnologies.com/order/catalog/product/AM6050] ** Labeled concentration = 1ug/ul *2 tubes for RNA sample and 1 tube with no RNA (replace with H2O) as negative control {| {{table}} | align="center" style="background:#f0f0f0;"|''' Components ''' | align="center" style="background:#f0f0f0;"|''' Volume ''' |- | RNA||0.5 ul (0.5 ug) |- | d(T)23VN (50 Β΅M)||2 ul |- | H2O||5.5 ul |- | Total||8 ul |} * Denatured RNA for 5 min at 70C and then put on ice * Added the following to tubes {| {{table}} | align="center" style="background:#f0f0f0;"|''' Components ''' | align="center" style="background:#f0f0f0;"|''' Volume ''' |- | M-MuLV Reaction Mix||10 ul |- | M-MuLV Enzyme Mix||2 ul |- | Total||20 ul |} * Incubated for one hour at 42C * Inactivated enzyme for 5 min at 80C *Purified cDNA in one Qiagen PCR clean up column and NTC in another **Eluted 30ul each *Nanodrop w/ dsDNA setting: **cDNA: 30.7 ng/ul **Neg Ctrl: 10.2 ng/ul ===Calculate Probes Needed=== ====V4==== {| {{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 |} ====V7==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Probe:target''' | align="center" style="background:#f0f0f0;"|'''1000:1''' | align="center" style="background:#f0f0f0;"|'''''' |- | Probe size||2486||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 (2486, 150nt)||1.21388894x10^8||g/mol |- | Amount Probe req'd||18.7||ng |} ===Probes, Target and Ampligase Buffer Mix=== *gDNA: 12878 (80.3ng/ul) *First strand cDNA from HBRR *[[Matt:LabNotes/2014-11-19#Final_Probes_to_Order:_Trim_to_12.2C000_probes | CA12k_Nov2014 Probes]] **V4 819nM x (150nt*325Da/nt + 79Da) x 10^-6 L/ul = 40.0 ng/ul **V7 768nM x (150nt*325Da/nt + 79Da) x 10^-6 L/ul = 37.5 ng/ul ***Diluted 1:2 with H2O for easier pipetting (final conc 20.0ng/ul and 18.75ng/ul) *NTC is the NTC from first strand cDNA synthesis *5nM of each supp oligos (5 x 10^-6 nmol/ul) ***100-fold more of each suppressor oligo = 1.5385 x 10^-14 mol = 15 x 10^-6 nmol {| {{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;"|'''5nM Supp Oligos''' | align="center" style="background:#f0f0f0;"|'''10X Ampligase Buffer''' | align="center" style="background:#f0f0f0;"|'''H2O''' | align="center" style="background:#f0f0f0;"|'''Total''' |- | 1||V4 - gDNA||1.3||3.8||3||3||18.9||30 |- | 2||V4 - cDNA||1.3||15||3||3||7.7||30 |- | 3||V4 - NTC||1.3||15||3||3||7.7||30 |- | 4||V7 - gDNA||1||3.8||3||3||19.2||30 |- | 5||V7 - cDNA||1||15||3||3||8||30 |- | 6||V7 - NTC||1||15||3||3||8||30 |} *Sample 4, accidentally added an extra 9ul H2O so added 4ul 10X Ampligase Buffer **Added 4ul of AmpLigase enzyme mix as well (vs 3ul for others) *Add 40ul mineral oil on top '''Program'''<br> * 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 20h * -> add 3ul AmpLigase enzyme mix (0.5U/ul AmpLigase in 1X AmpLigase buffer) * -> 55 C 20h-> 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 |- | 5||2||ISB_CA_AF||ISB_CA_AR.T2 |- | 6||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;"|'''6.5X Volume''' |- | Captured template||1||0 |- | 10uM Forward Primer||0.4||2.6 |- | 10uM Reverse Primer||0.4||0 |- | 2X KAPA SYBG MM||12.5||81.25 |- | H2O||10.7||69.55 |- | Total||25||153.4 |} *Aliquot 23.6ul from 6.5X 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)x15 -> 72C 3min [[File:20150501_CA12kNov2014_invitroPCRtest.JPG | 650px]] *Looks good; NTC are both negative so I won't amplify on next PCR ====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)x13 -> 72C 3min [[File:20150502_CA12kNov2014_invitroPCR.JPG|650px]] *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:2015-05-02_CA12kNov2014_invitro_gelcheck.jpg|450px]] *Labeled tubes and put in "Non-quantified Sequencing Libraries" **Sample1: MC20150502_CA12kNov14supp_V4gDNA-1 **Sample2: MC20150502_CA12kNov14supp_V4cDNA-2 **Sample3: MC20150502_CA12kNov14supp_V7gDNA-4 **Sample4: MC20150502_CA12kNov14supp_V7cDNA-5
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