Arturo Casini:Protocols

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PCR (2 annealing temperatures)
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

M9 medium (supplemented)
mix in an 1 liter glass bottle:

(all these solutions have to have been already sterilised)

Thiamine cannot be autoclaved, so it has to be sterilised with a 0.2 um filter

volume	       solution		stock concentration		final concentration in 1 liter

733.8 ml	water			water

200 ml		M9 salts		5X

34 ml		Thiamine		10 mg/ml			1 mM

10 ml		glycerol		40%				0.4%

20 ml		Casamino Acids	       10%				0.2%

2 ml		MgSO4		       1M				2 mM

200 ul		CaCl2			0.5 M				0.1 mM

total volume = 1 liter

cover with foil, don't autoclave, Thiamine is sensible to light and heat

put 400mg of thiamine powder in 40 ml of ddH2O, then filter with 0.2 um filter

<!-- ==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.-->

E. coli CaCl2 competent cell protocol
From Jan, Imperial College Biochemistry department

reference: http://www.med.nyu.edu/medicine/labs/blaserlab/Protocols/E-coli_competent_cells_protocol_&_transformation.pdf

Day 1

0. ALWAYS autoclave 0.1M CaCl2 and 0.1M CaCl2+15% glycerol solutions (can be autoclaved multiple times, so make up several runs’ worth), and 50ml tubes

1. Plate the cells on appropriate media and grow O/N @ 37°C.

Day 2

2. Inoculate a single colony into 100ml LB in a 250ml flask in the morning.

Or

2. Inoculate a single colony into 5ml LB in a 50ml falcon tube in the evening. Grow O/N @ 37°C.

3. Use 1ml to inoculate 100ml of LB in a 250ml flask the next morning.

Then…

4. Put appropriate centrifuge rotor in the cold room!

5. Shake @ 37°C for 1.5-3hrs, until OD600 is ~0.6.

6. Put the cells on ice for 10mins (keep cold from now on) – set the centrifuge to pre-cool if the option is available

7. Collect the cells by centrifugation in the big centrifuge (Thermo Sorvall RC6 Plus) for 5mins @ 4500rpm and -4°C. Make sure to have properly balanced the tubes to +/- 0.01 grams.

8. Discard supernatant and gently resuspend pellet on 10 ml cold 0.1M CaCl2 (Resuspend the CaCl2 before use). Cells are susceptible to mechanical disruption, so treat them nicely.

9. Incubate on ice for 20mins

10. Centrifuge for 5mins @ 4500rpm and -4°C

11. Discard supernatant and gently resuspend pellet in 5ml cold 0.1M CaCl2+15% glycerol

12. Dispense in microtubes (200μL/tube) on dry ice, or freeze in liquid N2.

13. Store at -80°C (and send the CaCl2 solutions for autoclave again if there’s enough left for another run)

E. coli chemical transformation
used on Stratagene XL1-Blue and cells made competent with the CaCl2 competent cell protocol

note: these cells are VERY fragile, so don't shake them or stress them in any way

do everything near a flame

always do a negative control with no DNA (skip step 6)

you can use refrozen cells for this

1) take cells from -80 and thaw them on ice

2) put 14 ml Falcon tubes (ROUND BOTTOM) on ice

3) put an LB flask/tube in water bath at 42 degs

you need 1 ml per transformation

do not let the container be completely submerged in water, or water from water bath might contaminate LB

4) take around 25 ul from cells' tube when they're de-frozen enough to be pipettable and put them in the Falcons.

Do not clip the caps.

volume of cells taken depends on how many transformations you're doing: a tube contains 200 ul, and has to be used completely, it can't be refrozen. 25 ul means up to 8 transformations.

5) incubate on ice for 10 mins

6) add DNA that you wish to transform into the cells

2.5 ul if it's assembly products, 1 ul if it's miniprep

7) incubate 30 mins on ice

8) give the cells a 45 secs heat shock in the waterbath at 42 degs, then put them back on ice for 2 mins

be VERY PRECISE here

9) add 0.9 ml (900 ul) LB and incubate in shaking incubator at 37 degs for 1 hour.

Keep the Falcons upright

10) transfer the colture to a 1.5 tube (total volume should be 1 ml)

11) give them a centrifuge blast: 13000 rpm for 1 sec, and discard most supernatant

12) resuspend ALL the cells in the remaining supernatant

13) take the plates with the appropriate antibiotic and label them, then transfer all the liquid from the 1.5 tubes to them (each tube its own plate). Spread the liquid all over the plate with an hockey stick.

14) place in an incubator at 37 degs overnight, agar side up.

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