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Baylor-exon-Sept09 (11/28)
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===Baylor (BCM) Creation=== '''PCR''' 1x 48x Template 0.4ul 19.2ul 2x Kapa Master Mix 50ul 50ul H2O 50ul 50ul eMIP-CA1F (100uM) 0.4ul 0.4ul eMIP-CA1R (100uM) 0.4ul 0.4ul 95C, 30sec -> (95C, 3sec -> 54C, 30sec -> 60C, 20sec -> plate read) x 25 -> 60C, 2min -> 15C hold '''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 ==Nicking Enzymes== '''First Round''' For each 50ul sample, add: -6ul 10x NEBuffer2 -5ul Nt.A1wI enzyme Incubate at 37C for 2hrs -> 4C hold *Run another Qiaquick purification '''Second Round''' For each 50ul sample, add: -6ul 10x NEBuffer2 -5ul Nb.BsrDI enzyme Incubate at 65C for 2hrs -> 4C hold *Run another Qiaquick purification -> Elute with 50ul H2O ==Size Selection== '''Preparation''' I split each sample in half (50ul -> 2x 25ul) and filled the remaining volume (25ul) with water. 50ul of 2x TBU Buffer was then added. The ladders were created by adding: -1ul 10bp ladder -9ul H2O -10ul 2x TBU Buffer Once the TBU gel was prepped, the samples were incubated at 75C for ~7min, then put on ice for ~1min. The ladders and samples were then loaded into the gel. After running the gels for 20min at 250V, they were stained with SYBR Gold. '''UV Cutting and Imaging''' [[Image:11_28_BCM_1.jpg]] ''Gel Image 1'' [[Image:11_28_BCM_2.jpg]] ''Gel Image 2'' '''Extraction''' I excised the bands at 100bp and sheared them at 14k rpm for 1:30. 400ul 1x TE solution was then added to each tube (8 tubes total) of sheared gel and the resulting mixture was incubated/vortexed at 37C for about 1 hour. The liquid remains were spun through a NanoSep column, which was then put through ethanol purification. Each tube was then eluted with 5ul water. When pooled, the total volume of BCM sample was roughly 40ul. I took this new (11/28) BCM sample and added it to the previous (11/19) BCM sample. I then ran another QIAquick purification to combine all the product and augment the concentration. The final product was eluted with 24ul water (enough to run multiple quantifications on). ==Quantification== Gel image: [[image:BCM_Quantification.jpg]] The sample was quantified via three methods. First I used a Qubit Flourometer, then a NanoDrop reading, then a gel quantification was run. To be safe, the conservative concentration was used. Results (all in ng/ul): Qubit NanoDrop Gel BCM 40.0 45.0 15.2 Using the gel quantification, the final product was 20ul with a concentration of 15ng/ul
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