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


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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