BisFirstExon
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bisFirstExon (bis) Creation
PCR
1x 48x Template 0.4ul 19.2ul 2x Kapa Master Mix 50ul 50ul H2O 50ul 50ul AP1V41U (100uM) 0.4ul 0.4ul AP2V4 (100uM) 0.4ul 0.4ul 95C, 30sec -> (95C, 3sec -> 60C, 50sec -> plate read) x 25 -> 60C, 2min -> 15C hold
- Note: Amplification stated at ~12 cycles
Ethanol Precipitation
Collected 24 reactions in a 16mL tube and added: 0.1x 3M NaOAc (240ul) 2.5x 100% EtOH (6mL) 8ul GlycoBlue -> Allow to cool in -70C freezer for at least 20 minutes -> Spin in 4C at 4k rpm for 20min -> Remove all liquid -> Suspend palette in 750ul 75% EtOH -> Move solution from 16mL tube to fresh 1.6mL tube -> Spin in 4C at 10k rpm for 5min -> Remove all liquid, allow palette to dry -> Suspend palette in 100ul H2O
Qiaquick Purification
Per tube of oligo solution: -Add 5x PB buffer -Mix in 1.6mL tubes and transfer solution to Qiaquick columns -Spin at 14k rpm for 1min -Toss supernatant -Add 750ul PE buffer to column -Spin at 14k rpm for 1min -Toss supernatant -Spin again, toss collection tube -Transfer column to fresh 1.6mL tube -Elute with 50ul H2O per column -Spin at 14k rpm for 1:30 -Toss Qiaquick column
Enzymatic Digestion
DpnII Enzyme
For each 50ul sample, add: -2ul DpnII (50U/ul) enzyme -5ul DpnII 10x Buffer -Incubate at 37C for 2hrs -> 4C hold
USER Enzyme
For each ~60ul sample, add: -4ul USER enzyme -Incubate at 37C for 2hrs -> 4C hold
- Run another Qiaquick purification
Nanodrop Readings
CES Tube 1: 169.3 ng/ul * 50ul = 8.5ug CES Tube 2: 148.7 ng/ul * 50ul = 7.4ug