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=== Production PCR === * '''V4 pool for all four sets (12 reaction for each set)''' {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="background:#f0f0f0;"|'''49x rxn mix''' |- | 20nM oligo pool||0.20||0.00 |- | 2x Kapa SYBG qPCR MM||50.00||2,450.00 |- | 100uM pAP1V41U||0.40||19.60 |- | 100uM AP2V4||0.40||19.60 |- | 2mM aa-dUTP||3.00||147.00 |- | H2O||46.00||2,254.00 |- | Total volume (ul)||100.00||4,900.00 |} - Aliquot 1197.6 ul in 1.5 mL tube, add 2.4ul of each oligo pool to each tube -> aliquot 100ul of reaction mix into strip tube<br> '''Program''' 95C 30sec -> (95C 3sec -> 55C 30sec-> 72C 20sec) x 16 -> 72C 2min -> 15C hold.<br> #1-V4 -> 16 cycles #2-V4 -> 16 cycles #3-V4 -> 14 cycles #4-V4 -> 15 cycles (Note: take the average of the qPCR result) [[File:2012_05_14_Probe-V4-pool.png| 500px]] *'''V6 pool for all four sets (12 reaction for each set)''' {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="background:#f0f0f0;"|'''49x rxn mix''' |- | 20nM oligo pool||0.20||0.00 |- | 2x Kapa SYBG qPCR MM||50.00||2,450.00 |- | 100uM pAP1V61U||0.40||19.60 |- | 100uM AP2V6||0.40||19.60 |- | 2mM aa-dUTP||3.00||147.00 |- | H2O||46.00||2,254.00 |- | Total volume (ul)||100.00||4,900.00 |} - Aliquot 1197.6 ul in 1.5 mL tube, add 2.4ul of each oligo pool to each tube -> aliquot 100ul of reaction mix into strip tube<br> '''Program''' 95C 30sec -> (95C 3sec -> 55C 30sec-> 72C 20sec) x 15 -> 72C 2min -> 15C hold.<br> #1-V6 -> 15 cycles #2-V6 -> 14 cycles #3-V6 -> 14 cycles #4-V6 -> 14 cycles (Note: take the average of the qPCR result) [[File:2012_05_14_Probe-V6-pool.png| 500px]] - Ethanol precipitation. - Pool amplified amplicons (total volume 1,200ul each set) into 15mL tube - Add 4ul of Glycoblue - Add 120ul of 3M NaoAc pH5.5 - Add 3mL of 100% EtOH - Put the tubes at -80C for 1hr - Centrifuge at 3,000rmp for 30min, 4C - Remove spnt. add 600ul of 75% EtOH and transfer the DNA pellet to 1.5 mL tube - Spin at 14,000rmp for 5min - Dry the pellet in the hood and resuspend with 100ul H2O - Re-purified each amplicon with two Qiaquick columns, eluted with 40ul EB => total volume 120ul each. - Measure concentration with Nanodrop. Probe IDs Conc. (ng/ul) Yields in 120ul (ug) #1-V4 83.70 10.04 #2-V4 125.10 15.01 #3-V4 110.10 13.21 #4-V4 133.80 16.06 #1-V6 122.50 14.70 #2-V6 106.20 12.74 #3-V6 132.20 15.86 #4-V6 101.70 12.20
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