Molecular Biology protocols, utilities and oddities
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Chamical transformation, DMSO+PEG (Chung & Miller, 1988 modified by Walhout et al., 2003)
This protocol was published by Walhout et al., "GATEWAY recombinational cloning: application to the cloning of large numbers of open reading frames or ORFeomes." Methods Enzymol. 2000;328:575-92.
It is a modification of C. T. Chung and R. H. Miller, "A rapid and convenient method for the preparation and storage of competent bacterial cells." Nucleic Acids Res. 1988;16:3580 (free at PMC).
Although the original publication by Walhout et al. is not free, you can find the original page explaining the transformation technique in Google Books, here. But I give tips too!
This is a very quick and effective transformation method. I effortlessly get efficiencies over 10^7 CFU/ug, which are not as high as commercial competent cells get, and not sufficient for library construction, but are surely good enough for everyday cloning.
I've seen people spend hours to wash, and wash, and wash big bottles of cell pellets to prepare electrocompetent cells, never testing their efficiency. If you're one of them, do a test - if your efficiency is lower around 10^7, give this protocol a try, it's incomparably quicker.
LB Broth pH 6.1 (10 g/l Tryptone, 5 g/l Yeast extract, 10 g/l NaCl - but adjust to pH 6.1 before autoclaving)
PEG:  10%   (average MW 3,350), e.g. Sigma P-4338 (but it also works with PEG-8000).
DMSO: 5%
Mg2+:  20 mM  (10 mM MgCl2 + 10 mM MgSO4)
   Procedure: To the LB add 10% solid PEG, the MgCl2  MgSO4 (these last two, both from 1M stocks) and mix to dissolve the PEG. Sterilize through a 0.22um syringe filter. Add the DMSO.
  1. Grow the host overnight in 5ml of LB, preferably from a single colony of a recently-streaked plate (that's good microbiology practice). Use antibiotic selection when appropriate.
  2. In the morning, use 500 ul of the overnight culture to inoculate 50 ml of LB (use an antibiotic only if the cells already bear a plasmid, to maintain selective pressure for it). Grow at 37°C and 180-200RPM until the OD600 reaches 0.3-0.6.
    I like to wait for an OD600 of 0.5-0.6, which will take about three hours. It'll take more time if the culture isn't thoroughly aerated (i.e. if your shaker isn't orbital).
  3. Centrifuge, 1000g, 10', 4°C.
    From this moment on, the cells must never be allowed to warm up. This is crucial.
  4. Remove the supernatant and ressuspend gently in 5ml of cold TSB.
    You might want to use a plastic Pasteur pipette to do the job. If it takes time, pause every minute to chill the tube again on ice.
  5. Leave the cells for 10 mins on ice.
  6. Aliquot the cells (250-350ul) and store at -80°C.
    I usually pre-chill the eppendorf tubes on ice (the normal stuff, 0°C), add the cells, and immediately snap-freeze them in dry ice. If you want to pre-chill the tubes in dry ice, leave them open, because they will get brittle - the lid might break loose when you open them to add the cells.
    You'll need 100ul per transformation. You can re-freeze cels once, with some loss of efficiency. In my case, 250 or 350 ul aliquots are handy enough.

   You should now determine the transformation efficiency of your new batch of cells.

KCM 5x:
KCl  0.5 M
CaCl2  0.15 M
MgCl2  0.25 M
(Store at -20°C, up to 6 months)
LB, pH 7.0 (the usual 10/5/10)
Tryptone         10 g/l
Yeast Extract    5 g/l
NaCl                10 g/l
Adjust to pH 7.0, adjust final volume and autoclave.
Can be stored at RT or +4°C for a couple of months.
LB-agar plates
Add 16 to 20g/l bacteriological agar to the LB broth, before autoclaving. Add a magnetic stirring bar too.
After autoclaving, leave in a 55°C waterbath for half an hour, or on a stirring plate until you can hold the bottle with your hands without feeling the urge to let it go. (This should be less than 60°C, or 95°C if you're a cooking-savvy grandma. In the latter case, the test is invalid). Then add the appropriate antibiotic, stir to mix, and plate.
Leaving the plates open for a couple of hours under a sterile hood will dry them a bit and help to absorb the transformation volume you'll plate on them. On the other side, if you want to let them solidify with the lid on, then plate them on a pile (one on top of the other), so that water will condense heavily only on the top lid of the pile.
Store plates sealed, at 4°C (time depends on the antibiotic; I use Amp plates within two weeks, Kanamycin and Chloramphenicol within a month).
  1. Take an aliquot of competent cells and let them thaw on ice
    (Seriously! Don't let them warm up! I mean it!)
    (Ok, you're a busy scientist. Then, you can hold the tube in your hands just enough to bring the plastic from -80°C to about 0°C, ok? Promise?)
  2. Chill on ice one eppendorf tube per transformation, and add:
  3.    H2O         70ul
       Ligation   10ul
       KCM 5x    20ul
       Total      100ul
    If you're using a supercoiled plasmid, just substitute ligation volume with plasmid DNA (picograms to nanograms) and water to 80ul, plus 20ul of KCM 5x.
    If you need to add more ligation volume, try to stay at or below 15 ul, to avoid reducing the transformation efficiency (adjust water accordingly.) Adding 20 ul of ligation is a bad idea.
  4. When the tube is cold, add 100ul of competent cells and flick gently to mix.
  5. Using a (wider-bore) 1ml pipet tip is preferrable.
  6. Leave 20 mins on ice
  7. Leave 10 mins at RT
  8. Add 600ul SOC (Wiki) or LB and incubate for 45 mins at 37°C
    SOC medium might improve transformation efficiency over LB.
    I usually add 400ul and not 600ul, to get more colonies in the 50ul plates.
  9. Plate, dispersing the liquid throughout the surface of the plate. Transfer to the incubator only when the plates are dry.
  10. How much volume, could you ask? With ligations, I usually set two plates per transformation: in one, I add 50ul of the transformation. Then I quickly spin down the rest (1', 6000rpm), discard 500ul of supernatant, ressuspend the pellet in what's left of the LB (~50ul), and spread it all on the second plate. The original protocol was to plate 50ul, 150ul and spin down all the rest (350ul), but 150ul can really take quite some time to dry.