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==Experimental Plan (See Noi's notes for actual data)== <!-- (1) Aliquot the seed oligos in the master tube, and perform expansion PCR on one small aliquot (Noi). (3/6) (2) Perform production PCR followed by nicking enzyme digestion and PAGE size selection (Noi, Matt) (3/7-3/8) (3) Prepare human gDNA, total RNA, and double-stranded cDNA for capture. gDNA can be from any human cells, such as the Hapmap sample. Total RNA should be the human brain reference RNA (get an aliquot from Rui). cDNA need to be prepared from total RNA through first strand synthesis (NEB E6300S) and second-strand synthesis (NEB E6111S). Please order both kits immediately. (Matt) (3/7-3/8) (4) Perform padlock capture on the three templates (gDNA, total RNA, ds-cDNA). Note that this probe set has no gap, so you only need Ampligase, but no Stoffel and dNTP. After exo digestion, proceed with PCR. You will need to design PCR primers that can anneal to the common linkers, and contain Illumina adaptors on the 5’ends. The common linker could be too short for forward and reverse primers. In case you can’t design good primers, please look into the barcodes. There are only four different sequences at the end of the barcodes, so you can simply design one common forward primers, and four reverse primers matching the barcodes. (Noi, Matt) (3/11-3/15) (5) In addition to quantifying the capturing efficiencies, we also want to quantify the abundance of different probes in the initial oligo pool. For this, design two additional primers with Illumina adaptors on the 5’-ends, and eMIP_CA1/eMIP_CA2 on the 3’-ends. Use this primer pair to amplify a small aliquot of the original seed oligos for Illumina sequencing. (Noi, Matt) (3/18-3/22) --> ===Oligo Resuspension=== * After receiving the probes, resuspend with H2O to the concentration 20 nM (in case it is lyophilized) ===Expansion PCR=== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="background:#f0f0f0;"|'''Final conc.''' |- | 20nM CustomArray Inc Oligoes||10.00||1nM |- | eMIP_CA1_F (100uM)||0.80||400nM |- | eMIP_CA1_R (100uM)||0.80||400nM |- | 2x Kapa SYBG MM||100.00||1x |- | H2O||88.40|| |- | Total||200.00|| |} '''Program'''<br> 95C 30sec -> (95C 5sec -> 52C 60sec-> 72C 30sec) x '''5''' -> (95C 5sec -> 60C 30sec-> 72C 30sec) x '''10-12''' -> 72C 2min -> 15C hold * Primer info. ** eMIP_CA1_F: TGCCT'''AGGACCGGATCAACT''' (TGCCT overhang), Tm of the short one = 53.06C, the long one = 63.28C ** eMIP_CA1_R: GAGCT'''TCGGTTCACGCAATG''' (GAGCT overhang), Tm of the short one = 54.41C, the long one = 62.95C * Note: ** The first five cycles, Tm of each primer was calculated without 5 nt overhang, then the Tm was increased based on the Tm of the full length primer ** Monitor number of cycles with real-time thermalcycler to prevent over-amplification. Terminate reactions right before the amplification curve plateaus * Purify with Qiaquick column (2 columns) and elute with EB buffer volume 50ul each --> total volume = 100ul * Measure DNA conc. with Nanodrop: __ * Dilute 1st round amplicon to 10nM for use as the template for the future amplification ===Production PCR=== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1 rxn''' | align="center" style="background:#f0f0f0;"|'''32x rxn mix''' |- | 1st round amplicons (10nM)||0.20||6.40 |- | eMIP_CA1_F (100uM)||0.40||12.80 |- | eMIP_CA1_R (100uM)||0.40||12.80 |- | 2x Kapa SYBG MM||50.00||1600.00 |- | H2O||49.00||1568.00 |- | Total||100.00||3200.00 |} '''Program'''<br> 95C 30sec -> (95C 5sec -> 60C 30sec-> 72C 20sec) x '''13''' -> 72C 2min -> 15C hold * Monitor cycles to prevent overamplification * Check the oligos by running 2ul before amplification in 6% TBU gel and quantify conc. if it corresponds to conc. specified on the tube? * Perform EtOH precipitation of the 32 rxn PCR in 4x 15mL tube ** 800ul amplified amplicon +80ul 3M NaOAc+2.6ul Glycoblue and 2mL 100EtOH ** incubate at -80C for 30min ** centrifuge at 3,000 rpm for 30 min at 4C ** discard supernatant and add 800ul cold 75% EtOH ** transfer DNA pellet to 1.5 mL tube (using pre-cut 1mL pipette tip) ** spin at 14,000 rpm for 5 min ** discard supernatant and air-dry pellet for 5 min in hood ** resuspend DNA with 150ul H20 ** measure conc. with Nanodrop:__ *Re-purify amplified-amplicon with Qiaquick column? ** Pool purified amplicons into one tube and measure DNA conc with Nanodrop:__ ===Nicking endonuclease digestion=== * Incubate 4ug of amplified amplicon with 10ul of Nt.AlwI (10U/ul) in total volume 200ul at 37C for 1hr (4x reactions?) * Heat inactivate enzyme at 80C for 20 min * Add 10ul Nb.BsrDI (10U/ul) at 65C for 1 hr * Purify with Qiaquick colums, elute with 50ul EB buffer each * Measure the conc. of nicked DNA with Nanodrop:__ ===PAGE size selection=== * Before doing PAGE size selection to purify the 170nt band, Run gel to check if DNA nicking worked well by loading the same amount of DNA before and after incubation with each enzyme at the same amount? * After EtOH precipatition and washing, resuspend the probe with 15ul each tube * Measure the final concentration: ===Sample Prep=== * gDNA: HapMap samples * total RNA: human brain reference samples ** 1 ug total in 33 ul * ds cDNA: First and second strand synthesis from 1 ug total RNA ** __ ug total in 33 ul, ====First Strand cDNA synthesis==== * Follow NEB E6300S protocol: [https://www.neb.com/protocols/1/01/01/first-strand-cdna-synthesis-e6300] Starting with 1ul of 1ug/ul total RNA brain reference * Mix components in microfuge tube {| {{table}} | align="center" style="background:#f0f0f0;"|''' Components ''' | align="center" style="background:#f0f0f0;"|''' Volume ''' |- | RNA||1 ul (1 ug) |- | d(T)23VN (50 μM)||2 ul |- | H2O||5 ul |- | Total||8 ul |} * Denature RNA for 5 min at 70C and then put on ice * Add the following to tube {| {{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 |} * Incubate for one hour at 42C * Inactivate enzyme for 5 min at 80C * Store at -20C Lab Notes: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-3-8 ====Second Strand cDNA synthesis==== *Follow NEB E6111S protocol: [https://www.neb.com/protocols/1/01/01/second-strand-cdna-synthesis-e6111] Starting with 20 ul cDNA (10-100 ng?) *Put in ice *Add 48 ul H20 *Add 8ul 10x Second strand synthesis reaction buffer *Add 4ul Second strand synthesis enzyme mix *Mix by pipetting *Incubate for 2.5 hours at 16C *Purify using Qiagen minelute column (elute 23ul) ===Padlock Probe Capture=== *[[Noi/NOTES/2013-3-12|Lab Procedures on Noi's Notes]] ====Design PCR primers==== {| {{table}} | align="center" style="background:#f0f0f0;"|''' Primer Name ''' | align="center" style="background:#f0f0f0;"|''' Sequence ''' | align="center" style="background:#f0f0f0;"|''' Tm ''' |- | ISB_CA_AF||AATGATACGGCGACCACCGAGATCTACACGCCTGCATATCGGGAAGCTGAAG||80.27 |- | ISB_CA_AR.T1||CAAGCAGAAGACGGCATACGAGATCGTGATCGGTCTGCCTTCCCGATATCCGACGG||81.72 |- | ISB_CA_AR.T2||CAAGCAGAAGACGGCATACGAGATACATCGCGGTCTGCCTTCCCGATATCCGACGG||81.82 |- | ISB_CA_AR.T3||CAAGCAGAAGACGGCATACGAGATGCCTAACGGTCTGCCTTCCCGATATCCGACGG||81.90 |- | ISB_CA_RP1||TACACGCCTGCATATCGGGAAGCTGAAG||70.61 |- | ISB_CA_RP2v1||CGGTCTGCCTTCCCGATATCCGACGG||72.49 |- | ISB_CA_IRPv1||CCGTCGGATATCGGGAAGGCAGACCG||72.49 |- | ISB_CA_AF.T1||AATGATACGGCGACCACCGAGATCCGTGATTACACGCCTGCATATCGGGAAGCTGAAG||81.46 |- | ISB_CA_AF.T2||AATGATACGGCGACCACCGAGATCACATCGTACACGCCTGCATATCGGGAAGCTGAAG||81.40 |- | ISB_CA_AF.T3||AATGATACGGCGACCACCGAGATCGCCTAATACACGCCTGCATATCGGGAAGCTGAAG||81.40 |- | ISB_CA_AR.L1||CAAGCAGAAGACGGCATACGAGATCGGTCTGCCACGCCGACGGACGCTA||83.00 |- | ISB_CA_AR.L2||CAAGCAGAAGACGGCATACGAGATCGGTCTGCCACGCCGACGGAGAACTTG||81.92 |- | ISB_CA_AR.L3||CAAGCAGAAGACGGCATACGAGATCGGTCTGCCACGCCGACGGCTACTTCG||82.69 |- | ISB_CA_AR.L4||CAAGCAGAAGACGGCATACGAGATCGGTCTGCCACGCCGACGGGACGAA||83.16 |- | ISB_CA_AR.L5||CAAGCAGAAGACGGCATACGAGATCGGTCTGCCACGCCGACGGTCTTGCG||83.28 |- | ISB_CA_AR.L6||CAAGCAGAAGACGGCATACGAGATCGGTCTGCCACGCCGACGGAACGGT||83.27 |- | ISB_CA_AR.L7||CAAGCAGAAGACGGCATACGAGATCGGTCTGCCACGCCGACGGCTGGTT||83.46 |- | ISB_CA_AR.L8||CAAGCAGAAGACGGCATACGAGATCGGTCTGCCACGCCGACGGTGTATCGC||82.79 |- | ISB_CA_AR.L9||CAAGCAGAAGACGGCATACGAGATCGGTCTGCCACGCCGACGGCGTATCG||83.04 |- | ISB_CA_RP2v2||CGGTCTGCCACGCCGACGG||71.38 |- | ISB_CA_IRPv2||AATGATACGGCGACCACCGAGATC||67.10 |} [[Media:InSituProbePrimerDesign3-12-13v2.docx|Primer Design Details]] ===Quantify abundance of initial probes=== ISB_CA_QPF: AATGATACGGCGACCACCGAGATC CGATCAG TGCCTAGGACCGGATCAACT Tm = 80.81 <br> ISB_CA_QPR: CAAGCAGAAGACGGCATACGAGAT TACACGT GAGCTTCGGTTCACGCAATG Tm = 78.80 <br> ISB_CA_QPR1 (Read 1 Primer): CGATCAG TGCCTAGGACCGGATCAACT Tm = 69.86 <br> ISB_CA_QPR2 (Read 2 Primer): TACACGT GAGCTTCGGTTCACGCAATG Tm = 69.96 <br>
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