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*Both rounds of Nicking Enzymes, used on both BCM sets, failed to properly digest the DNA. In order to get a better understanding of the situation, the BCM set was re-amplified and the two enzymes, Nt.A1wI and Nb.BsrDI were tested as follows: ==BCM Preparation== '''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 *Note: After the QIAquick purification, I attempted to use the NanoDrop to roughly estimate the amount of DNA. Unfortunately, I was unable to acquire any data as the NanoDrop machine was unable to process the sample. A gel was run to ensure that there was in fact sample in the mixture, which the gel confirmed: [[Image:3 sets.jpg]] From here 20ul were taken from the main tube of BCM solution (which originally contained 100ul) and were split into 2 tubes of 10ul solution. To each tube, 40ul water was added to make a total volume of 50ul for each tube. ==Nt.A1wI== Using one of the 50ul tubes, I added: 10ul 2x NEBuffer2 35ul H2O 3ul Nt.A1wI enzyme The reagents were incubated for 2 hours at 37C ==Nb.BsrDI== Using the other 50ul tube, I added: 2ul Nb.BsrDI enzyme The reagents were incubated for 2 hours at 65C ==Results== After running both enzymes through size-selection gels, it is clear that the Nb.BsrDI enzyme is '''NOT''' working properly: [[image:Nt.A1wI_Nb.BsrDI_test.jpg]] *Concluded, after checking the theoretical matches, there was an ''experimental'' error. It was determined that the Nb.BsrDI enzyme needed NEBuffer 2 before being incubated, which I failed to do. After testing the Nb.BsrDI enzyme with buffer, the results were clear: [[image:Nb.BsrDI_test.jpg]] Note: Gel appears to have a problem, but is sufficient for examining effects of enzyme Nb.BsrDI Clearly both enzymes work, the problem was in the experimental setup. After review of the NEB website on the activity of the enzymes I re-calibrated the numbers: For both enzymes, 1 unit can digest 1ug of DNA -> First measure amount of DNA via NanoDrop -> If necessary, split sample into 2 tubes and fill up to 50ul with water -> add 6ul 10x NEBuffer 2 -> add 2ul Nt.A1wI (or Nb.BsrDI) at 10U/ul -> incubate at correct temperature (37C or 65C, respectively) for 2 hours -> hold at 4C ==Real-Time Test== As a final test, I ran another half-plate of BCM (11/19) and removed 6ul of the sample after running the Nt.A1wI and after running the Nb.BsrDI enzymes. Below is a gel image used to quantify as well as compare efficacy of the two enzymes: [[image:Enzyme_Test_Quant.jpg]] ==Conclusion== Because the labeled amount of Enzyme:DNA was calibrated using lambda DNA, which can be considered, for our purposes, randomly assembled, the chances that an enzyme would find the correct 4-mer cutting site is 1/512. This means that the company's enzyme is calibrated for a much lower frequency cutting than for our purposes (which is a guaranteed 1/probe cutting frequency), we have to augment the amount of enzyme used. In this case, for each ug of DNA to be digested we will need roughly 4-5 times the recommended. I used 2x the recommended amount of enzyme, which as the gel showed is not enough. Math note: From a theoretical standpoint, the enzyme is calibrated for a ratio of 1:512 cutting sites:DNA length. Our probes have a ratio of 1:140 which is roughly 3.65x more frequent. Still, given the solution that the enzymes rest in, over-estimating the amount of enzyme needed is a safe move.
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