CES36k18bp
CES36k18bp (CES) Creation[edit]
PCR
1x 48x Template 0.4ul 19.2ul 2x Kapa Master Mix 50ul 50ul H2O 50ul 50ul AP1V61U (100uM) 0.4ul 19.2ul AP2V6 (100uM) 0.4ul 19.2ul 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[edit]
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
Size Selection and Extraction[edit]
Size Selection
Based on the Nanodrop readings, I used this set as a test for how to divide all the sets. I divided each tube of 50ul into two tubes of 25ul and added 25ul 2x TBU Buffer, making 50ul total for each gel. The samples were run against a 10bp ladder. Each gel was then cut to excise the band at ~100bp. The first gels run were slightly overloaded, but the second gels were clean:
First set of CES gels
Second set of CES gels
After excising the correct band from the gel, each band was cut in half. These halves were placed into shearing tubes (a 0.5mL tube with a hold poked through the bottom resting freely inside a 1.6mL tube) and spun down at 14,000 rpm for 1:30.
Extraction
After the gel fragments were sheared, the 0.5mL tubes were tossed and 400ul of 1x TE Buffer was added to each tube. Each tube was then placed in a 37C incubator and placed on a spinner/vortexer for approximately 1 hour. After this each tube was run through another round of ethanol purification. The ethanol products were suspended in 5ul of H2O and collected into a fresh 1.6mL tube.
Quantification[edit]
Gel Images:
File:Bis BCM CES Quantification1.jpg
Gel image 1
File:Bis BCM CES Quantification2.jpg
Gel image 2
Each sample was quantified via two methods. First I used a Qubit Flourometer, then two different gel quantifications were run. To be safe, the conservative concentrations were used.
Results (all in ng/ul): Qubit Gel 1 Gel 2 Average (Between Gels) bis 30.8 6.62 4.54 5.60 CES 32.5 6.89 4.40 5.60 BCM 3.84 2.9 1.26 2.10