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==CA12k_Nov2014 V4 and V7 Probe Production== *Repeat [[Matt:LabNotes/2015-1-5]] ===Production PCR=== **[[Matt:LabNotes/2015-1-5#Expansion_PCR | 10nM 1st round amplicons]] *V4 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_V4 (10nM)||0.2||10 |- | 2X KAPA SYBG MM||50||2500 |- | AP1V4U (100uM)||0.4||20 |- | AP2V4 (100uM)||0.4||20 |- | H2O||49||2450 |- | Total||100||5000 |} *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 15sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold [[File: | 800px]] ===EtOH Precipitation=== *6 15-ml tubes (with 8 wells of PCR product each) for V4 and 6 tubes 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 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 50ul H2O ===Qia Column Purification=== *Purified in 6 columns following Qiagen protocol *Eluted each column with 50ul and combined all V4 probes into one 1.5ml and all V7 probes into another *Measured concentration with Nanodrop: ~150ul of V4 probes: 156.5 ng/ul => ~23.5 ug ~150ul of V7 probes: 163.6 ng/ul => ~24.5 ug ===Lambda Exonuclease Digestion=== *Divide each into 3 pcr tubes of 50ul with total amplicon of <10ug each {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | Amplicon||50 |- | 10X Lambda Exo Buffer||10 |- | Lambda Exonuclease||10 |- | H2O||30 |- | Total||100 |} *Incubated at 37C for 1hr *Purified with 3 Zymo ssDNA/RNA columns each *Eluted with 40ul each *Recombined and measured ssDNA with Nanodrop: **V4: 77.0ng/ul x 120ul = 9.24ug (78.6% yield) **V7: 61.0ng/ul x 120ul = 7.32ug (59.8% yield) ===Remove Amplification Adapters=== ====USER==== *Split each set into 2 PCR tubes and add 5ul USER {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | SS-amplicon||60 |- | USER||5 |- | H2O||20 |- | Total||85 |} *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||10 |- | 100uM RE-DpnII guide oligo||5 |} RE-DpnII guide oligo: V4: RE DpnII V4 V7: RE DpnII V7 *Incubated at 94C for 2 min, then 37C for 3 min *Added 5ul DpnII *Incubated at 37C for overnight ===TBU Gel: Digestion Check=== *Combine into 1 tube for each set *2ul Digested Sample + 2ul 2X Urea Loading Dye *5ul Undigested V7 10nM 1st round amplicon) + 5ul 2X Urea Loading Dye *2X (0.5ul Low Mass Ladder + 1.5ul H2O + 2ul 2X Urea Loading Dye) [[File:2015-01-20_V4V7_Digest_GelCheck.jpg]] *V7 Undigested (middle lane) is extremely faint but can see band around 200bp *Not sure why V4 lane is squiggly but can see the 3 bands *V7 lane shows only 2 bands but both are below the undigested V7 band ===Zymo Column Purification=== *2 columns for each set *Eluted 30ul each column (60ul total) *Nanodrop **V4: 78.8ng/ul x 60ul = 4.7ug **V7: 38.8ng/ul x 60ul = 2.3ug ===PAGE Size Selection=== *Run 2 gels for V4 and 1 gel for V7 **200V for 30min {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''2X Volume''' |- | V4 Probes||60 |- | TBE-Urea Buffer 2X||60 |} {| {{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||3 |- | TBE-Urea Buffer 2X||15 |- | H2O||12 |- | Total||30 |} *V4 Gel 1 [[File:2015-01-20_SizeSelect_V4_Gel1.jpg]] *V4 Gel 2 [[File:2015-01-20_SizeSelect_V4_Gel2.jpg]] *V7 Gel 1 [[File:2015-01-20_SizeSelect_V7_Gel1.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 6 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 (~450ul per tube except for 1 V4 tube which spilled) *Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 45ul of 3M NaoAc pH 5.2 *Vortexed and placed the 6 tubes at -80C for overnight *Spun 6 tubes at 10,000rpm at 4C for 30min *Discard supernatant and add 650ul cold 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 for V4 and 20ul for V7) ===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 |- | |} *V4 **2ul sample lane was loaded poorly so was not used in concentration calculations [[File:2015-01-24_V4_GelQuant.jpg|650px]] *V7 **Only use ladder bands 100bp, 200bp, and 400bp [[File:2015-01-21_V7_GelQuant.jpg|650px]] *V4: 65.3 ng/ul => 65.3 ng/ul / (150nt*325Da/nt + 79Da) = 1337nM (38ul) *V7: 39.2 ng/ul => 39.2 ng/ul / (150nt*325Da/nt + 79Da) = 803nM (18ul) [[Media:2015-01-21_V7_GelQuant.xlsx | V7 Concentration calculation]] [[Media:2015-01-24_V4_GelQuant.xlsx | V4 Concentration calculation]]
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