Team:CSU Fort Collins/NoteBook/Aug
8.8.16 Running gel with PCR products of 7.29.16 Looks good 8.9.16 Performed the gel purification protocol from the lab, including the following changes: Only did 1 membrane wash Did not pre heat the elution buffer Did the final elution with 15uL of the 10mM tris-HCL ph 8.8 Both parts of the gene block will be nanodropped tomorrow to see how high the concentration of DNA is 8.10.16 PCR with minipreped stataclone vector + insert from culture started on 7.29.16 We have 4 samples, 1A, 1B, 2B, 2C Two different annealing temperatures for each sample. 68 & 72 So we will have 8 PCR reactions. For C. Limon 1 Component 50μl reaction Molecular grade H2O Added first. 28 μL 5x GC Buffer 10μL 10 mM dNTPs 1.0μL L. synthase forward (10μM) 2.5μL C. Limon 1 or 2 middle primer (10μM) 2.5μL Template DNA 1.0μL Tom’s Phusion DNA polymerase Added last 5uL So we’re going to need (4)(2.5μL) of each primer. In the end we want (10μL)(10μM) so.. dilute(10μL)(10μM) = 10-4μmol = 10-4μmol(1L/25μmol) = 4*10-6L = 4μL stock 4μL stock + 6μL H2O = 10μL(10μM) primers & 8μL of dNTP’s stock 25 mM We want (8μL)(10mM) Dilute (8μL)(10mM) = 8*10-5mmol = 8*10-5mmol (1L/25mmol) = 3.2*10-6L = 3.2μL 3.2μL + 4.8μL H2O = 8μL(10mM) dNTP’s For C. Limon 2 Component 50μl reaction Molecular grade H2O Added first. 32.5μL 5x GC Buffer 10μL 10 mM dNTPs 1.0μL LS p2(10μM) 2.5μL C. Limon 2 Forward (10μM) 2.5μL Template DNA 1.0μL Tom’s Phusion DNA polymerase Added last 5uL So we’re going to need (4)(2.5μL) of each primer. Cycle Step 3-step protocol Cycles Temp (oC) Time Initial denaturation 98 5 min 1 Denaturation 98 10s Extension 68-72 (gradient) 1.5 min 35 Hold 4 hold 8.10.16 -Ran a gel with the completed C.Limon PCR products listed above from 8.9.16, results are shown below: ladder, next three were sharon’s stuff, 1A-68, 1B-68, 2B-68, 2C-68, 1A-72, 1B-72, 2B-72, 2C-72 -Nanodropped C. Sten 1 & 2 gel purification products results are shown below: C. Sten 1 green tube- 9.9 ng/ul, 260/280- 2.07 C. Sten 1 blue tube- 16.2 ng/ul, 260/280-1.92 C. Sten 2 orange tube- 14.1 ng/ul, 260/280-1.84 C. Sten 2 yellow tube- 17.4 ng/ul, 260/280-1.72 8.12.2016 Fluorimetry on C. limon gel purification calibration : 99.76 Concentration(ng/uL) Error 1A-68: 18.9k -1.172 1A-72: 76.58 6.4 1B-68: 53.96 -2.9 1B-72: 33.34 -2.472 2B-68: 27.93 -5.44 2B-72: 69.96 -10.76 2C-68: 76.63 ? 2C-72 : 13.27 -11.73 8.13.16 -We tried infusion cloning with C.sten 1 and 2 g-blocks, these were the ones that were not in the strataclone vector, so after PCR amplification and running a gel to confirm amplification, we attempted to just directly clone both g-blocks into the pLC71 vector We used a 1:1:1 molar ratio for 1st g-block:2nd g-block:pLC71 and the values for each are shown below, keep in mind we had two PCR products for each g-block, so they are separated by eppendorf tube color, in this case we only used C.sten 1 blue and C.sten 2 yellow C.Sten 1 blue: 1.36 uL C. Sten 2 yellow: 1.02uL pLC71:0.88uL, dilution- 2uL pLC71 + 2uL sterile H2O, this way we could use 1.8uL instead of 0.88 which is hard to achieve with a pipette 3.8uL of water We did a negative control that contained everything listed above, except for the infusion mix -After following the Infusion protocol, we followed the Interlab transformation protocol, except we added 2 uL of the plasmid instead of 1uL, also when we plated after the outgrowth period, we use 50uL on one plate, and the rest on another plate, ~200uL
