"
Team:Washington/Protocols
From 2013.igem.org
Workflow:
- iGEM Vector Information
- General Cloning Strategy
- 1) Isolation of Plasmid DNA (miniprep)
- 2) General PCR Protocol (also see PCR GoTaq - product (30-200ng/ ul) Check on gel PCR Purification and/or Dpnl Digest
- 3) General Digestion Protocol Check on gel PCR Purification or Heat Inactivation (check enzyme temp and time, usually 80C for 20min)
- 4) Agarose Gel Electrophoresis
- 5) General Ligation Protocol (Don’t forget background control plates) Heat Inactivation (optional - up to 10 fold increase) - 65° for 10 minutes
- 6) Heat shock/chemical competent transformation
- 7) Colony PCR with Green taq Miniprep (stocks can be made from this culture - add 1ml extra)
- 8) DNA Sequencing
- 9) Making Glycerol Frozen Stocks
Light Sensor Protocols
To carry out a typical Green light inducible GFP experiment:
1. Transform plasmids containing pLPCB and pJT119B operon to a competent cell. Our lab uses JT2 (Tabor, J. J. et al., Multichromatic Control of Gene Expression in Escherichia coli, J. Mol. Biol. (2010), doi:10.1016/j.jmb.2010.10.03) cells for gfp output testing.
2. Plate cells on LB agar plates with XXX and XXX antibiotics. See table below for list of systems and plasmids with their required antibiotics.
3. After incubation at 37 oC overnight, pick a single colony, and inoculate 5 mL of LB medium in a 50 mL Falcon tube.
4. Grow cultures to saturation overnight at 37 oC with shaking.
5. Determine cell density (O.D.600 measurement), and dilute overnight cultures to a final O.D.600 of 1x10-4 in M9 minimal medium supplemented with casamino acids
(link: http://openwetware.org/wiki/M9_salts 5x m9salts)
(link: http://openwetware.org/wiki/Endy:M9_media/supplemented m9 supplemented)and appropriate antibiotics.
For 96 well plates:
1. Pipette 260 ul of the inoculated M9 + Casamino acid media
(link: http://openwetware.org/wiki/M9_salts 5x m9salts)
(link: http://openwetware.org/wiki/Endy:M9_media/supplemented into each well of interest in a clear bottomed black 96 well plate (Supplier information -- maybe even the name).
2. Cover the plate with breathable tape.
3. Orient the 96 well plate on the tablet such that the wells line up with the appropriate light source on the the tablet.
4. Secure the 96 well plate on top of the tablet with tape.
5. Incubate tablet and 96 well plate on an orbital shaker (RPM?) for 8 hours at 37 oC.
6. Remove 200 ul of culture from each well, and pipette them to 96 well plates for analysis (Highlight analysis and link to pertinent protocol).
96 well analysis (GFP expression):
1. GFP expression levels are analyzed on a spectramax m5e plate reader (bottom read) with an excitation of 480 nm, and emission at 520nm. Set the plate reader to automix the samples for 5 seconds prior to analysis.
2. GFP expression levels were normalized against cell density by dividing the relative fluorescence unit values by O.D.600 data.
For 60mm mini petridish:
1. Add 7.5ml of the inoculatted M9+cassamino acid media into the plate which is labelled for green light testing.
2. Add another 7.5ml of the inoculated media and a put it into another plate which is labelled as dark for the dark condition test.
3. Foil the mini petri dish for the dark ones.
4. Get another clear 60mm mini petri dish and then use it as a spacer plate from the tablet to the testing petri dish which will sit on top of the green light. This will prevent overheating of the culture. (refer to supplemental image)
5. Tape both spacer and testing petri dish ontop of the testing tablet securely and put them on top of the orbital shaker in the 37C incubator.
6. After eight hours, take 200ul of the culture which has been growing on top of the shaker and pipette them to 96well plates for RFU plate reading
7. We took the Optical density of the cells with lambda 600nm
8. Also took endpoint fluorescence which is excitation= 480nm, emission= 520nm, autocutoff= 515nm. Automixed 5seconds before reading.
9. Then we normalized RFU(relative fluorescence unit) with the optical density by dividing the RFU with the OD.
