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Baylor-exon-Sept09 (11/4)
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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 *Note: Amplification started after ~13 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 ==Nicking Enzymes== *NOTE: These enzymes were ''fresh'', having arrived only on 11/6/09. However, I was not able to proceed with the enzymatic digestion until 11/15/09. '''First Round''' For each 50ul sample, add: -10ul 10x NEBuffer2 -35ul H2O -3ul Nt.A1wI enzyme Incubate at 37C for 2hrs -> 4C hold *Run another Qiaquick purification '''Second Round''' For each 50ul sample, add: -2ul Nb.BsrDI enzyme Incubate at 65C for 2hrs -> 4C hold *Run another Qiaquick purification -> Elute with 50ul H2O ==Size Selection== '''Preparation''' I decided to skip the NanoDrop quantification and split each tube in half, yielding 4x 25ul samples. Added 25ul water and 50ul 2x TBU Buffer to each tube, making a total volume of 100ul. 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. Upon viewing the gels through a UV stage, it was '''again''' evident that the enzymatic digestion '''did not''' work: '''UV Cutting and Imaging''' [[Image:BCM_2_SizeSelection_1.jpg]] ''Gel Image 1'' [[Image:BCM_2_SizeSelection_2.jpg]] ''Gel Image 2: Extracted band at 120bp'' *Note: I decided to continue with half of the gel content excised at 120 to be compared with the ''CES'' and ''bis'' sets
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