Arturo Casini:Protocols
2 temperature steps PCR
to amplify miniprepped plasmid DNA using primers that have tails
mix in small 0.2 ml thin walled PCR strip tubes
mix in this order
Distilled H20 18.75 ul
Barnes buffer (B'S on the tube) 2.5 ul
Forward primer (100 ng/ul) 1 ul (4 ng / ul)
Reverse primer (100 ng/ul) 1 ul (4 ng / ul)
Template DNA (miniprep) 1 ul (0.2 ng/ul)
dNTP mix (10 mM) 0.5 ul
PfuUltra II fusion DNApol 0,25 ul
TOTAL 25 ul
1x
95 degs, 30 secs
10x
95 degs, 30 secs
annealing portion Tm -3 degs, 30 secs (if more than one, the lowest)
68 degs, 15 secs + 15 secs for each kb
20x
95 degs, 30 secs
whole primer Tm -3 degs, 30 secs (if more than one, the lowest)
68 degs, 15 secs + 15 secs for each kb
1x
68 degs, 2 mins
1x
4 degs, infinite
Cover lid temperature: 105-110 degs
Plate reader measurements for promoter characterisation
Tested on E.coli strains Rosetta 2 DE3 pLyss, Top10, Mg1655
Tested on M9 and LB
M9 media has 0.4 % glycerol and 1:1000 Kanamycin (and also 1:1000 Cloramphenicol for Rosetta 2 cultures)
Use BMG Labtech Polarstar Omega
Use Greiner 96 well flat bottom microplates.
Use a plastic lid.
Use M9 as blank, same volume as the cultures (no need to add Chloramphenicol here)
Use 2 spacers under the optics in the machine.
Keep the lid on the plate while shaking, but remove it while measuring, so to improve measurement accuracy and limit oxygen supply problems.
Put 100 ul liquid in the wells, and set path length correction at 100 ul in ARTURO OD protocol.
Make sure to check the layout in the protocols before using them.
Make sure not to change the gain once is set for the full series of experiments on a given plate.
Make sure not to change the layout for the full series of experiments on a given plate.
Set the plate reader incubator at 37 degs.
ARTURO GFP-RFP protocol is set on measuring these fluorescences:
GFP excitation: 485
GFP emission: 510
RFP excitation: 587
RFP emission: 610
When modifying any protocol, always check the timing and make sure you have enough time to run ARTURO OD, ARTURO GFP-RFP and ARTURO SHAKE within 30 mins leaving some time to start the protocols and moving the lid.
ARTURO SHAKE shakes at 100 rpm.
When measuring the OD of the overnight cultures, make sure that the cells are fully suspended before pipetting.
When measuring the OD of the overnight cultures use a p200 to take the sample out, try not to touch the inside of the tube and wipe the pipette with ethanol after each use to avoid cross contamination.
When measuring the OD of the overnight cultures add M9 to dilute AFTER adding the cultures to the cuvettes, and mix them by pipetting (no need to add Chloramphenicol here).
Take the measurement right after mixing to avoid settling of the cells. Samples 1-6 go in bays 2-7.
Day before:
Start overnight liquid cultures in 14 ml clip top tubes.
Use 5 ml M9 (add 1:1000 Chloramphenicol if using Rosetta2)
If you didn't start these culture from a replica plate, make a replica plate now.
Day of the experiment:
1) Prepare and label 1.5 ml tubes with 0.5 ml M9 (add 1:1000 Chloramphenicol if using Rosetta2)
2) Measure the OD600 of the overnight cultures diluting 1:5 (200 ul culture, 800 ul M9, no need to add Chloramphenicol here). Use 1000 ul M9 as blank (no need to add Chloramphenicol here)
3) Add some of the overnight cultures in the prepared 1.5 ml tubes. Use this formula to calculate the volume to add:
(desired OD600 / overnight culture OD600) * ml of new culture = ml of overnight culture to add
which in this protocol is (0.1 / overnight culture OD600) * 5 = ml of overnight culture to add
Make sure to resuspend the overnight cultures by vortexing before pipetting.
4) Take 100 ul from the 1.5 ml tubes and load the microplate wells. Use 100 ul M9 as blank, and make sure to have around 6 blank wells.
5) Take measurements every 30 mins using ARTURO OD and ARTURO GFP-RFP protocols. Remember to always take the measurements without the lid on.
At timepoint 00:00 start the timer when you start the ARTURO OD protocol, for the following timepoints start the ARTURO OD protocol when the 30 mins timer rings, and immediately reset it.
After taking the measurements put the lid back on and start the ARTURO SHAKE protocol.
