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==CA12k_Nov2014_V7 Probe Production== *Production of Padlock Probes targeting constitutive exon cDNA (V7 primers) of [[Matt:LabNotes/2014-11-19#Assembly_of_oligo_pool_for_ordering]] ===Production PCR=== *[[Matt:LabNotes/2015-1-5#Expansion_PCR | 10nM 1st round amplicons]] *V7 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_V7 (10nM)||0.2||10 |- | 2X KAPA SYBG MM||50||2500 |- | AP1V7U (100uM)||0.4||20 |- | AP2V7 (100uM)||0.4||20 |- | H2O||49||2450 |- | Total||100||5000 |} '''Program''' 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold [[File:011214_CA12kNov14_ProductionPCR_V7.JPG | 800px]] ===EtOH Precipitation=== *6 15-ml tubes (with 8 wells of PCR product each) for V7 **800ul PCR product **2000ul 100% EtOH **2.7ul GlycoBlue **80ul 3M NaOAc pH 5.2-5.5 *Vortexed and put in -80C for overnight *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 6 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 6 tubes of 100ul in 6 columns following Qiagen protocol *Eluted each column with 50ul and combined all into one 1.5ml *Measured concentration with Nanodrop: ~290ul of V7 probes: 93.2 ng/ul => ~27 ug ===Lambda Exonuclease Digestion=== *Divide each into 3 pcr tubes of ~100ul with total amplicon of <10ug each {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | Amplicon||100 |- | 10X Lambda Exo Buffer||12.5 |- | Lambda Exonuclease||12.5 |- | Total||125 |} *Incubated at 37C for 1hr *Purified with 3 Zymo ssDNA/RNA columns *Eluted with 40ul each *Recombined and measured ssDNA with Nanodrop: **62.9ng/ul x 120ul = 7.5ug (56% yield) ===Remove Amplification Adapters=== ====USER==== *Save 2ul for gel check later *Split into 2 PCR tubes and add 5ul USER {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | SS-amplicon||59 |- | USER||5 |- | 10X DpnII Buffer||8 |- | H2O||8 |- | Total||80 |} *Incubate at 37C for 2.5 hours ====DpnII==== *Added 15ul of the following {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X DpnII Buffer||2 |- | 100uM RE-DpnII_V7 guide oligo||5 |- | H2O||8 |- |} *Incubated at 94C for 2 min, then 37C for 3 min *Added 5ul DpnII *Incubated at 37C for overnight (~15hrs) ===TBU Gel: Digestion Check=== *Combine into 1 tube *2ul Sample + 2ul 2X Urea Loading Dye *0.5ul Low Mass Ladder + 1.5ul H2O + 2ul 2X Urea Loading Dye [[File:2015-01-14_V7_Digest_GelCheck.jpg]] *Gel ran poorly for some reason but I don't have anymore undigested probe to compare to (unless I want to waste 5ul of 10nM 1st rnd amplicon) so I will just check the gel when I do size selection **The band to cut out should be 150bp so almost halfway between 200bp and 100bp ladder bands ===Zymo Column Purification=== *Eluted 30ul each column (60ul total) *Nanodrop **41.7ng/ul x 60ul = 2.5ug (oddly low again like [[Matt:LabNotes/2015-1-5#Zymo_Column_Purification | last time]]) ***This time 33% yield which is better than 25% yield last time ===PAGE Size Selection=== *Run 1 gel for V7 **220V for 30min {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1X Volume''' |- | V7 Probes||60 |- | TBE-Urea Buffer 2X||60 |} {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | Low Mass Ladder||1 |- | TBE-Urea Buffer 2X||5 |- | H2O||4 |- | Total||10 |} [[File:2015-01-14_SizeSelect_V7.jpg]] ===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 2 Nanosep columns 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 2 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 2 tubes at -80C for overnight *Spun 2 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 combine ===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 |- | |} [[File:2015-01-15_V7_GelQuant.jpg|650px]] *Only use ladder bands 100bp, 200bp, and 400bp to get trendline with better R^2 *28 ng/ul => 28 ng/ul / (150nt*325Da/nt + 79Da) = 573nM (18ul) [[Media:2015-01-15_V7_GelQuant.xlsx | How I calculated concentration]]
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