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==CA12k_Nov2014 RevComp Probe Production== *Production of Padlock Probes targeting cDNA (V6 and V8 primers) of [[Matt:LabNotes/2014-11-19#Assembly_of_oligo_pool_for_ordering]] ===Expansion PCR=== *[[Matt:LabNotes/2014-12-18]] conclusion: 21 cycles of PCR starting with 100nM seed oligos *Make 2 tubes: **V6 primers - CA12k_Nov2014_Contig_RevComp **V8 primers - CA12k_Nov2014_Const_RevComp {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''Contig of Exons RevComp(V6)''' | align="center" style="background:#f0f0f0;"|'''Constitutive Exons RevComp(V8)''' |- | CA12k_Nov2014 Seed Oligos (900nM)||15||15 |- | 2X KAPA SYBG MM||67.5||67.5 |- | 100uM AP1V6U||0.6||0 |- | 100uM AP2V6||0.6||0 |- | 100uM AP1V8U||0||0.6 |- | 100uM AP2V8||0||0.6 |- | H2O||51.3||51.3 |- | Total||135||135 |} '''Program''' 95C 30sec -> (95C 5sec -> 55C 30sec-> 72C 45sec) x 21 -> 72C 2min -> 15C hold [[File:012614_CA12kNov14_ExpansionPCR_V6V8.JPG|650px]] *Did not think V6 amplified correctly (possibly due to underpipetting a primer) but [[Matt:LabNotes/2015-1-5#Expansion_PCR|V4]] showed a similar curve **Nanodrop also shows V6 amplified as much or more than V4 *Purified each with 1 Qiagen column and eluted with 50ul H2O *Nanodrop: **V6: 11.8 ng/ul -> 11.8 ng/ul / (188bp*660Da/bp) = 95nM **V8: 16.2 ng/ul -> 16.2 ng/ul / (190bp*660Da/bp) = 129nM *Diluted to 10nM **V6: 48ul x 95nM = '''456ul''' x 10nM ***Add 408ul H2O **V8: 48ul x 129nM = '''619ul''' x 10nM ***Add 571ul H2O ====Gel Check==== *Use 5ul of 10nM expansion PCR'd probes + 5ul 2X Loading dye *Use 2ul 10nM Agi26k0gap expansion PCR'd probes as positive control [[File:2015-01-26_1stRndAmplicon_V6V8_Gel.jpg]] *Apparently Nanodrop is not accurate enough at low conc *V6 is missing a band at 188bp... **Try Expansion PCR with V6 primers on the last of the seed oligos ====V6 Expansion PCR Try 2==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | CA12k_Nov2014 Seed Oligos (900nM)||14 |- | 2X KAPA SYBG MM||50 |- | 100uM AP1V6U||1 |- | 100uM AP2V6||1 |- | H2O||34 |- | Total||100 |} '''Program''' 95C 30sec -> (95C 5sec -> 55C 30sec-> 72C 45sec) x 30 -> 72C 2min -> 15C hold [[File:012614_CA12kNov14_ExpansionPCR_V6_try2.JPG|650px]] *Stopped after 22 cycles *Purified each with 1 Qiagen column and eluted with 50ul H2O *Nanodrop: **V6: 17.9 ng/ul -> 17.9 ng/ul / (188bp*660Da/bp) = 144nM *Diluted to 10nM **V6: 48ul x 144nM = '''691ul''' x 10nM ***Add 643ul H2O ===Production PCR=== *V6 master mix {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume (1X)''' | align="center" style="background:#f0f0f0;"|'''Volume (50X)''' |- | First round amplicon CA12k_Nov14_V6 (10nM)||0.2||10 |- | 2X KAPA SYBG MM||50||2500 |- | AP1V6U (100uM)||0.4||20 |- | AP2V6 (100uM)||0.4||20 |- | H2O||49||2450 |- | Total||100||5000 |} *V8 master mix {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume (1X)''' | align="center" style="background:#f0f0f0;"|'''Volume (50X)''' |- | First round amplicon CA12k_Nov14_V8 (10nM)||0.2||10 |- | 2X KAPA SYBG MM||50||2500 |- | AP1V8U (100uM)||0.4||20 |- | AP2V8 (100uM)||0.4||20 |- | H2O||49||2450 |- | Total||100||5000 |} '''Program''' 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 22 -> 72C 2min -> 15C hold *Stopped after 15 cycles [[File:012714_CA12kNov14_ProductionPCR_V6V8.JPG | 800px]] ===EtOH Precipitation=== *6 5-ml tubes (with 8 wells of PCR product each) for V6 and 6 tubes for V8 **800ul PCR product **2000ul 100% EtOH **2.7ul GlycoBlue **80ul 3M NaOAc pH 5.2-5.5 *Vortexed and put in -80C for 1hr *Centrifuged at 3000rpm at 4C for 30 min *Discarded supernatant and added 800ul of cold 80% EtOH *Transferred DNA pellet (with 1ml pipette tip) to 12 1.5mL tubes *Centrifuged 1.5ml tubes at 14,000rpm for 5 min at 4C *Discarded supernatant and air-dried for 5 min in hood *Resuspended DNA with 100ul H2O ===Qia Column Purification=== *Purified 12 tubes of 100ul in 12 columns following Qiagen protocol *Eluted each column with 50ul and combined all V6 probes into one 1.5ml and all V8 probes into another *Measured concentration with Nanodrop: ~290ul of V6 probes: 158.9 ng/ul => ~ 46 ug <br> ~290ul of V8 probes: 183.1 ng/ul => ~ 53 ug ===Lambda Exonuclease Digestion=== *Divide each into 6 pcr tubes of 48ul with total amplicon of <10ug each {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | Amplicon||48 |- | 10X Lambda Exo Buffer||10 |- | Lambda Exonuclease||10 |- | H2O||32 |- | Total||100 |} *Incubated at 37C for 1hr *Purified with 6 Zymo ssDNA/RNA columns *Eluted with 40ul each *Recombined and measured ssDNA with Nanodrop: **V6: 64.0ng/ul x 240ul = 15.4ug (67% yield) **V8: 65.0ng/ul x 240ul = 15.6ug (59% yield) ===Remove Amplification Adapters=== ====USER==== *Split each set into 3 PCR tubes and add 5ul USER {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | SS-amplicon||80 |- | USER||5 |- | Total||85 |} *Incubate at 37C for 3 hours ====DpnII==== *Added 15ul of the following {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X DpnII Buffer||10 |- | 100uM RE-DpnII guide oligo||5 |} RE-DpnII guide oligo: V6: RE DpnII V6 V8: RE DpnII V8 *Incubated at 94C for 2 min, then 37C for 3 min *Added 5ul DpnII *Incubated at 37C for overnight (16 hrs) ===TBU Gel: Digestion Check=== *Combine into 1 tube for each set *2ul Digested Sample + 2ul 2X Urea Loading Dye *5ul Undigested sample (10nM V8 1st round amplicon) + 5ul 2X Urea Loading Dye *0.5ul Low Mass Ladder + 1.5ul H2O + 2ul 2X Urea Loading Dye [[File:2015-01-28_V6V8_Digest_GelCheck.jpg]] ===Zymo Column Purification=== *3 columns for each set *Eluted 20ul each column (60ul total) *Nanodrop **V6: 137.3ng/ul x 60ul = 8.2ug **V8: 116.2ng/ul x 60ul = 6.9ug ===PAGE Size Selection=== *Run 2 gels for V6 and 2 gels for V8 **200V for 35min {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''2X Volume''' |- | V6 Probes||60 |- | H2O ||60 |- | TBE-Urea Buffer 2X||120 |} {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''2X Volume''' |- | V8 Probes||60 |- | H2O ||60 |- | TBE-Urea Buffer 2X||120 |} {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | Low Mass Ladder||4 |- | TBE-Urea Buffer 2X||20 |- | H2O||16 |- | Total||40 |} ===EtOH Precipitation=== *Put cut out gel in 0.5mL tube (with holes at bottom and centrifuged the tube at 15,000 rpm, for 3 min at RT. *Transfered the gel remaining in 0.5 mL tube to 2.0 mL tube below with pipette tip. *Added 900 ul of 1X TE buffer *Vortexed for 60 min at 37 C in incubator *Centrifuged at 15,000 rpm for 3 min at RT *Transfered the clear spnt. to 8 Nanosep column and centrifuged at 15,000 rpm for 3 min *Transfered the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min *Transfered spnt to fresh 1.5 mL tube (~400ul per tube) *Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 40ul of 3M NaoAc pH 5.2 *Vortexed and placed the 8 tubes at -80C for 30 min *Spun 8 tubes at 10,000rpm at 4C for 30min *Discard supernatant and add 650ul 80% EtOH and spin at 14,000rpm at 4C for 5min *Discard supernatant and let dry in hood for 10min *Resuspend each tube with 10ul and recombine (40ul total each) ===TBU Gel Quantification=== *Dilute 2ul to 10ul with H2O *Add 10ul 2X TBE-Urea Buffer *Load wells with 2, 4, 6, and 8 ul *Load Low Mass DNA ladders in 0.5, 1, and 2 ul *Ran at 200 V for 35min {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''0.5 ladder''' | align="center" style="background:#f0f0f0;"|'''1 ladder''' | align="center" style="background:#f0f0f0;"|''' 2 ul ''' | align="center" style="background:#f0f0f0;"|''' 4 ul ''' | align="center" style="background:#f0f0f0;"|''' 6 ul ''' | align="center" style="background:#f0f0f0;"|''' 8 ul ''' | align="center" style="background:#f0f0f0;"|'''2 ladder''' |- | DNA volume||0.5||1||0.1||0.2||0.3||0.4||2 |- | H2O||1.5||1||0.9||1.8||2.7||3.6||0 |- | 2X Buffer||2||2||1||2||3||4||2 |- | Total||4||4||2||4||6||8||4 |- | |} *V6 [[File:2015-01-30_V6_GelQuant.jpg|650px]] *V8 [[File:2015-01-30_V8_GelQuant.jpg|650px]] *Only use ladder bands 100bp, 200bp, and 400bp because others were too saturated and non-linear *V6: 41.7ng/ul => ng/ul / (150nt*325Da/nt + 79Da) = 854nM (38ul) *V8: 42.7ng/ul => ng/ul / (150nt*325Da/nt + 79Da) = 874nM (38ul) [[Media:2015-01-30_V6V7_GelQuant.xlsx | How I calculated concentration]]
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