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==CA12k_Nov2014_V4 Probe Production== *Production of Padlock Probes (V4) [[Matt:LabNotes/2014-11-19#Assembly_of_oligo_pool_for_ordering]] *Do double 96 100ul PCR Reactions ===Production PCR=== *[[Matt:LabNotes/2015-1-5#Expansion_PCR | 10nM 1st round amplicons]] *V4 master mix made in 2 5ml tubes {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume (1X)''' | align="center" style="background:#f0f0f0;"|'''Volume (100X)''' |- | First round amplicon CA12k_Nov14_V7 (10nM)||0.2||20 |- | 2X KAPA SYBG MM||50||5000 |- | AP1V4U (100uM)||0.4||40 |- | AP2V4 (100uM)||0.4||40 |- | H2O||49||4900 |- | Total||100||10000 |} '''Program''' 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold <br> [[File:032515_CA12kNov14_ProductionPCR_V4.JPG | 800px]] ===Zymo DNA Clean & Concentrator 100=== *Purify 10ml of PCR product with 1 Zymo-spin V Column/Reservoir **Add 50ml DNA Binding Buffer to 10ml product (Use 2 50ml tubes) *Elute with 540ul Elution buffer **Should elute with < 400ul next time because it won't all fit in column ***Lost about 80ul and ended with low yield (50ug normally after EtOH precipitation and Qia purification) *Measured concentration with Nanodrop: ~460ul of V4 probes: 71.3 ng/ul => 32.8ug ===Repeat Production PCR=== *[[Matt:LabNotes/2015-1-5#Expansion_PCR | 10nM 1st round amplicons]] *V4 master mix made in 1 15ml centrifugal tube *Poured into trough/reservoir and used multi-channel pipette to save time pipetting into pcr tubes {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume (1X)''' | align="center" style="background:#f0f0f0;"|'''Volume (100X)''' |- | First round amplicon CA12k_Nov14_V7 (10nM)||0.2||20 |- | 2X KAPA SYBG MM||50||5000 |- | AP1V4U (100uM)||0.4||40 |- | AP2V4 (100uM)||0.4||40 |- | H2O||49||4900 |- | Total||100||10000 |} '''Program''' 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold [[File:032615_CA12kNov14_ProductionPCR_V4.JPG | 800px]] ===Zymo DNA Clean & Concentrator 100=== *Tried using reservoir & multi-channel pipette removing product from 6 strips of pcr tubes but spilled ~1ml (product+binding buffer) pouring from reservoir into 50ml tubes *Purify 10ml of PCR product with 1 Zymo-spin V Column/Reservoir **Add 50ml DNA Binding Buffer to 10ml product (Use 2 50ml tubes) *Elute with 400ul H2O **Somehow final volume is greater than 400ul *Measured concentration with Nanodrop: ~410ul of V4 probes: 76.1 ng/ul => 31.2ug ====Potential Reasons for Low Yield==== *Second time I got 30ug when normal for EtOH precipitation method is 50ug *Possible reasons: **Potentially the vacuum is not strong enough to pull all solution through? (even though it's slow this is not likely) **Losing solution on the sides of the 50ml tubes used to mix binding buffer + amplicon (definitely happening but shouldn't be 40% of amplicon) **400ul elution volume still too much to allow all elution buffer to get out of column membrane when centrifuged *From Alan: Try heating up elution buffer or incubating the column with elution buffer at 37C for 5min ===Lambda Exonuclease Digestion=== *Combine 2 Production PCR products and Divide into 9 pcr tubes of ~110ul with total amplicon of <10ug each {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | Amplicon||96 |- | 10X Lambda Exo Buffer||12 |- | Lambda Exonuclease||12 |- | Total||120 |} *Incubated at 37C for 1hr *Purified with 9 Zymo ssDNA/RNA columns *Eluted with 25ul each *Recombined and measured ssDNA with Nanodrop: **100.7ng/ul x 225ul = 22.7ug (70.9% yield) ===Remove Amplification Adapters=== ====USER==== *Split into 4 PCR tubes and add 5ul USER {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | SS-amplicon||56 |- | USER||5 |- | 10X DpnII Buffer||8 |- | H2O||11 |- | Total||80 |} *Incubate at 37C for 2 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_V4 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) ===Zymo Column Purification=== *Eluted 30ul each column (115ul total) *Nanodrop **126.8ng/ul x 115ul = 14.6ug (66% yield) ===PAGE Size Selection=== *Run 3 gels (4 gels ideal but ran out of 2D TBU gels) **200V for 40min {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''3X Volume''' |- | V4 Probes||115 |- | TBE-Urea Buffer 2X||115 |} {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''3X Volume''' |- | Low Mass Ladder||3 |- | TBE-Urea Buffer 2X||15 |- | H2O||12 |- | Total||30 |} *Gel 1 [[File:2015-03-28_CA12kNov2014_V4_SizeSelect_Gel1.jpg]] *Gel 2 [[File:2015-03-28_CA12kNov14_V4_SizeSelect_Gel2.jpg]] *Gel 3 [[File:2015-03-28_CA12kNov14_V4_SizeSelect_Gel3.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. *Transferred 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 60min at 37 C in incubator *Centrifuged at 15,000 rpm for 3 min at RT *Transferred the clear spnt. to 6 Nanosep columns and centrifuged at 15,000 rpm for 3 min *Transferred the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min *Transferred spnt to 6 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 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 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:2015-03-30_CA12kNov14_V4_GelQuant.jpg|650px]] *Only use ladder bands 100bp, 200bp, to get trendline with better R^2 *70 ng/ul => 70 ng/ul / (150nt*325Da/nt + 79Da) = 1.43uM (1,433nM) (58ul) [[Media:2015-03-30_CA12kNov14_V4_GelQuant.xlsx | How I calculated concentration]]
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