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 Amplifying plasmids
A) Growth of Bacteria
To produce competent bacterias
1. Prepare Difco LB agar:
 40 g diluted in 1L of distillated water, without antibiotics.
2. Growth your “empty” bacterias in these plates at 37 ºC, for 12 h.
3. Prepare liquid Difco LB broth media
 250g diluted in 1L of distillated water. Autoclave for 15 min at 121 oC.
4. Select some colonies from LB agar and dissolve them in a sample of liquid LB
media.
5. Split the suspension in flasks containing 200 mL of liquid LB media.
6. Incubate your flasks at RT and start to collect samples 3 hours later and some times
after that to measure the OD (600 nm).
7. Stop culture when your culture reaches 0.4 OD (Note: this is the point that you have
the competent cells).
8. Centrifuge 3000 rpm, for 20 min, at 4 ºC.
9. Resuspend in 200 mL transformation buffer.
10.
Keep the suspensions in ice (20 min).
11.
Centrifuge 3000 rpm, 20 min, 4 ºC.
12.
Suspend in 12 mL (6%) transformation buffer + 8% DMSO.
13.
Froze your cells directly at -80 oC.
B) Inserting the plasmids into the bacteria
1. Thaw the competent bacteria but do not warm it.
2. Keep the bacteria in ice during the procedure (4 oC).
3. Split the bacteria in samples of 100 µL and add 0.1 µg of your plasmid (4 oC).
4. Keep the bacteria in ice (4 oC) for 40 min.
5. Put your samples at 42 oC for 30 sec to transfer de plasmids into the cells (“heat
chock”).
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6. Keep your samples in ice (4 oC) for 2 min.
7. Transfer each one of your samples to 1 mL of liquid LB media for 20 min at 37
o
C, 300 rpm shaker.
8. Distribute your bacterias in plates containing LB agar with antibiotics.
9. Incubate the plates at 37 oC for 12 h.
10. Take the colonies and grow up then in liquid LB media with antibiotics.
► Antibiotics:
Ampicillin Sodium Salt (easy to melt):
 Dissolve 500 mg in 10 ml of 35% ethanol. Prepare samples of 1 mL and
froze them at -20o C. Final concentration is 250g/mL (dilute stock 200x).
Karamicin:

Final concentration is 100 g/mL. Prepare Karamicin exactly ampicillin, at 10
mg/mL (dilute in water). Dilute 100x to use.
► Identification of plates:
Plates with Ampicillin: A (draw one line in each plate)
Plates with Karamicin: K (draw two lines in each plate)
Plates with LB only: LB (no lines)
C. Extraction of plasmids DNAs from bacterias
1. Use the QIAGEN Plasmid Midi Kits to purify your DNA (protocol from
manufacturer).
2. At the end, quantify your DNA measuring OD of your samples diluted 1:200 in
Milli-Q water. The result in g/l will be:
Concentration (g/l) = OD x 200 x 50
1000