IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/08/04

iGEM Project name 1

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BioFilm Track Obtained air-dried at room temperature 96-plates with biofilm on bottom of layer Stained wells using 0.1 w/v crystal violet stain (150ul added per well) Incubate for 15 mins at 37C to fixate stain Eluted well samples using 95 % ethanol and transfered onto 2nd 96 well plate Used OD reader to determine OD per well (Results and test specs on google docs) Examined Tube culture of biofilm (staph 8325) OD reading done after elution with 95% Ethanol, transfered 1ml of sample into cuvette per volume level of ethanol added Biofilm growth for Tube culture (5ml B2 used) Blank used: 1ml 95% Ethanol OD Readings Ethanol Added(ml) OD (at 600nm) 2ml 3.941 3ml 0.141 5ml 0.091 7ml 0.140 7ml 0.216 Phage Track Started UV induction Experiment Obtained ON culture [(staph 8325+5ml B2) grown in shaker (200rpm, 37C)] Transfered 100ul ON culture into 5ml B2 (x2) Incubated at 37C in incubator for 3hrs Centrifuged at 6000 g for 10mins at 25C Resuspended cells in 5ml 0.1 MGSO4 (placed in shaker for 15mins at 200 rpm, 37C) Transferred cells into sterile 25ml beakers Irradiated cells with constant swirling (RPM unknown, used hands) Irradiated culture (A) for 60 secs (short wave, 254 nm wave length) Irradiated 2nd culture (B) for 90 secs (short wave, 254nm) Irradiated both cultures for additional 15 secs on medium wave length (356nm) afterwards Transferred cell culture(along with 0.1M MgSO4 solution)back into 2 new sterile glass tubes. Incubated both culture tubes in 37 C incubator for 10mins before taking time zero reading OD Readings Time (min) OD CB (90sec) OD CA (60sec) 0 2.024 2.299 40 2.181 2.387 70 2.452 2.694 110 3.004 3.244 Blank sample used for reader: 500ul B2+500ul 0.1M MgSO4 (MgSO4 used to stay consistent with lysate content (B2+ 0.1M MgSO4) Used shaker after 40mins mark (200rpm, 37C) instead of static incubator. Refilled each culture with (500ul B2 + 500 ul 0.1M MgSO4 after each OD reading) to keep volume constant at 5ml Irradiated for an additional 60sec using 254nm shortwave length UV meter after 70mins mark (OD didn't go down) OD should have an decreasing trend for both cultures A and B (with B faster because longer dosage of UV); experiment didn't work; stored tubes at 4C Prepared 2 ON cultures of indicator strain RN4220, + 1 ON culture of staph 8325 for plaque assay, additional UV induction testing (1 RN4220 from plate, 1 from glycerol stock) All Placed in shaker at 8:30pm (200 rpm , 37C) QS Track PCR agrCA, agr C, and agr A (protein cds) Perform triplicate reactions for each of the above protein cds using a different colony from a plate containing Staph aureus NCTC 8325. Modified reaction conditions slightly from yesterday. Used more DMSO (6%) and changed annealing temperature to 62 °C. See below for PCR reagent table (without primers) PCR Master mix Reagent 1x rxn volume (uL) Master Mix 5x rxn buffer 5 x11 55 10mM dNTP 2 x11 22 sdH2O 5.55 x11 55.5 Phusion polymerase 0.05 x11 0.55 MgCl2 2 x10 20 DMSO 1.5 x10 15 Total 14.75 147.5 Primer pairs that enclose the protein cds were added at 1.25 μL forward and reverse primers for each 1 x reaction. Included water control. PCR Cycles: 98C @ 4min Cycle 27x: 98C @ 10 sec 62C @ 30 sec 72C @ 45 sec 72C @ 10 min 10C @ hold Gelled PCR products in 1.2% agarose, 0.5X TBE, GelStar stain in agaorse, for 45 minutes. Bands showed up for agr A and agr C the correct band size (~700 bp and ~1300 bp, respectively. Did not see a band for agr CA May be due to insufficient elongation time. Raise to 30 seconds/kb. For agr AC (2kb) this means elongating for 1 minute. Can also lower annealing temperature slightly to 60 °C. Perhaps the presence of DMSO lowered the Tm of the primers. However, the addition of Mg ions may have counteracted this effect. Raise number of cycles to 30. Doesn't increase overall PCR time by much anyway. Planning to PCR purify agr A and agr C. Not much volume of PCR product, so maybe combine the triplicates to salvage more DNA. The source of DNA may be from different colonies, but those colonies all came from the same source colony. dspB Track On old PCR samples, try both the iGEM and Biobrick RD and ligations to test out the different methods Vicki: iGEM method; Marianne: biobrick method Restriction Digest Vicki: Protocol: Restriction digest protocol in Protocol binder (SOP) Changes: incubation time = 1 hour instead of 2 hours Restriction Digest Supermix REAGENTS 1 RXN VOLUME (uL) SUPERMIX Buffer 2 5 x6 30 BSA 0.5 x6 3 ddH2O 37 x6 222 Total 42.5 255 DNA: add 5uL of each B, C, D, psb1c3 (b,c,d) Enzymes: EcoRI & PstI - add 1uL each to each tube Marianne: Protocol: RD protocol from biobrick assembly protocols RE supermix: same as the RE supermix above DNA: add 5uL of each A,E,F,psc1c3 (a,e,f) Enzymes: EcoRI & PstI; add 1uL each to each tube Changes: 37C for 30min stead of 15 min Concentration measurements Tube Concentration (ng/uL) A 99.6 B 65.0 C 104.7 D 115.6 E 115.1 F 131.1 a 20.3 b 17.5 c 22.6 Average of a-e = ~20ng/uL Ligations Vicki - Protocol: See ligation protocol from SOP Calculations [math]\displaystyle{ ratio \times \frac{insert length}{vector length} \times vector mass = insert mass (ng) }[/math] [math]\displaystyle{ 6 \times \frac{1200}{2400} \times 20 = 60 ng }[/math] [math]\displaystyle{ 1uL vector\times\frac{20ng}{1uL}\times\frac{60ng}{20ng}\times\frac{1uL}{x ng} = }[/math] Where x = concentration of insert Calcuations Tube Amount of vector(uL) Amount of insert (uL) Scale up (x5) Amount of vector (uL) Amount of insert A 1 0.602 x5 5 3.01 B 1 0.923 x5 5 4.615 C 1 0.573 x5 5 2.865 D 1 0.519 x5 5 2.595 E 1 0.517 x5 5 2.585 F 1 0.458 x5 5 2.29 Put ligation mixes B,C,D into tubes 1,2,3, respectively Put ligation mixes A,E,F into tubes 4,5,6, respectively Transformation Protocol: Transformation protocol from SOP Changes: 10uL of ligation mix instead of 1uL; 1 hour incubation instead of 2 hours Transforming ligation mixes 1,2,3,4,5,6 Marianne: Put plates 4,5,6 (A,E,F, respectively) in 37C incubator @ 1600 Vicki: Put plates 1,2,3 (B,C,D, respectively) in 37C incubator @ 1630 Vicki Ma 19:55, 10 August 2010 (EDT)