- 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
|Ethanol Added(ml)||OD (at 600nm)|
- 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
|Time (min)||OD CB (90sec) || OD CA (60sec) |
- 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)
- 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|
- 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.
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
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|
- 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
- Average of a-e = ~20ng/uL
Vicki - Protocol: See ligation protocol from SOP
- Where x = concentration of insert
|Tube||Amount of vector(uL)||Amount of insert (uL)||Scale up (x5)||Amount of vector (uL)||Amount of insert|
- Put ligation mixes B,C,D into tubes 1,2,3, respectively
- Put ligation mixes A,E,F into tubes 4,5,6, respectively
- 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)