IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/08/13

iGEM Project name 1

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dspB Track PCR off gDNA for more PCR products PCR Master mix Reagent 1x rxn volume (uL) Master Mix 5x rxn buffer 5 x7.5 37.5 10mM dNTP 1 x7.5 7.5 sdH2O 6.65 x7.5 49.875 Phusion polymerase 0.1 x7.5 .75 MgCl2 2 x7.5 15 DMSO - 5% 1.25 x7.5 9.375 Total 21 157.5 PCR Tubes Tube Content A1,A2,A3 dspB his B1,B2,B3 dspB no-his W (H2O control) To A1,A2,A3: add 2uL of up-his & dw primers each To B1,B2,B3: add 2uL of up & dw primers each PCR Cycles: 98C @ 3min Cycle 27x: 98C @ 10 sec 72C @ 30 sec 72C @ 40 sec 72C @ 10 min 10C @ hold Start: 0907 End: 1010 Gel verification colony PCR products Protocol: gel verification protocol in Protocol (SOP) Changes: 1% agarose gel Machine conditions: 0.5x TBE buffer, 100V, 45min Gel orientation: Gel orientation A1 A2 A3 100bp ladder B1 B2 B3 W (control) Results: O/N cultures 2 tubes (3T1, 4T3) taken out at 0950 Miniprep Miniprepped 3T1, 4T3 Protocol: in SOP binder (Alkaline lysis) Restriction Digest on miniprepped DNA See RD protocol (BioBricks) Gel Verification on mini-prepped RD Protocol: gel verification protocol in Protocol (SOP) Changes: 1% agarose gel Machine conditions: 0.5x TBE buffer, Gel orientation: Gel orientation 3T1 100bp ladder 4T3 chlor Results: Plan: RD + ligation + transformation Restriction Digest Protocol: Biobrick Protocol Restriction Digest Supermix REAGENTS 1 RXN VOLUME (uL) Vicki's SUPERMIX Buffer 2 5 x13 65 BSA 0.5 x13 6.5 ddH2O 37 x13 481 Total 42.5 552.5 DNA: add 5uL of backbone + 2uL of insert each Enzymes: EcoRI and SpeI (1uL each to each tube) Restriction Digest Supermix REAGENTS 1 RXN VOLUME (uL) Marianne's SUPERMIX Buffer 2 5 x33 165 BSA 0.5 x33 16.5 ddH2O 37 x33 1221 Total 42.5 1402.5 DNA: add 5uL of backbone + 2uL of insert each Enzymes: EcoRI and SpeI (1uL each to each tube) RD Tubes: AA, TA, CA, AB, TB, CB, 1-16 all contain insert dspB His AC, TC, CC< AD, TD, CD, 17-32 all contain insert dspB no-His Ligation Calculations [math]\displaystyle{ ratio \times \frac{insert \rm length}{vector \rm length} \times vector \rm mass = insert \rm mass (ng) }[/math] [math]\displaystyle{ 3 \times \frac{1200}{2400} \times = ng }[/math] [math]\displaystyle{ 1uL \rm vector\times\frac{ng \rm vector}{1uL vector}\times\frac{ng \rm insert}{ng \rm vector}\times\frac{uL \rm insert}{x ng \rm insert} = }[/math] Where x = concentration of insert Vicki: ligation - 6:1 ratio using amp backbone amp+tet backbone chlor backbone Calculations Tube Amount of vector(uL) Amount of insert (uL) A 3 2.559 B 3 2.808 C 3 2.820 D 3 3.042 Marianne: Ligation - chlor backbone using ratios: 2:1,3:1,4:1,6:1,7:1,8:1,9:1,10:1 Calculations for ratio 2:1 Tube Amount of vector(uL) Amount of insert (uL) A2 3 0.852 B2 3 0.936 C2 3 0.941 D2 3 1.014 Calculations for ratio 3:1 Tube Amount of vector(uL) Amount of insert (uL) A3 3 1.2801 B3 3 1.4046 C3 3 1.4106 D3 3 1.5216 Calculations for ratio 4:1 Tube Amount of vector(uL) Amount of insert (uL) A4 3 1.707 B4 3 1.872 C4 3 1.881 D4 3 2.028 Calculations for ratio 6:1 Tube Amount of vector(uL) Amount of insert (uL) A6 3 2.559 B6 3 2.808 C6 3 2.820 D6 3 3.042 Calculations for ratio 7:1 Tube Amount of vector(uL) Amount of insert (uL) A7 3 2.988 B7 3 3.279 C7 3 3.291 D7 3 3.552 Calculations for ratio 8:1 Tube Amount of vector(uL) Amount of insert (uL) A8 3 3.414 B8 3 3.747 C8 3 3.762 D8 3 4.059 Calculations for ratio 9:1 Tube Amount of vector(uL) Amount of insert (uL) A9 3 3.840 B9 3 4.215 C9 3 4.233 D9 3 4.566 Calculations for ratio 10:1 Tube Amount of vector(uL) Amount of insert (uL) A10 3 4.266 B10 3 4.583 C10 3 4.701 D10 3 5.073 Transformation Protocol: Transformation protocol from SOP Changes: 10uL of ligation mix instead of 1uL; 1 hour incubation instead of 2 hours Biofilm Track Eric F. + Melody 8325-4 Biofilm Growth Streaked a TSA plate with 8325-4 TSB O/N culture and put in incubator at 37°C RN4220 Biofilm Growth Completed Day 3 of the protocol for both plates Test #1 - control E5 showed growth Test #2 - controls B2, B11, C5, E7 and G2 showed growth We decided to continue the experiment as a wet run (due to the contamination the results are meaningless) Next time we will perform the inoculation step in the bio safety cabinet The 60°C air dry step was completed using a heat block For the overnight step, we placed the plates in the bio safety cabinet