Transforming Competent Cells with Plasmid DNA
Reference:
Protocol provided by PGC Scientifics Corporation.
Summary:
Transformation - the heritable modification of the properties of a competent bacterium by
DNA from another bacterial strain.
DH5 competent cells will be used (e.g., PGC Scientific). A plasmid with DNA
resistant to ampicillin from another bacterial strain (foreign DNA) will be introduced to
the DNA of the competent bacterium (host DNA) and streaked on LB agar plates with
antibiotic. Cells with the foreign DNA will be resistant to ampicillin, resulting in colonies
on LB agar with ampicillin. Cells without the foreign DNA will lyse!!
Procedure:
1. Remove cells from –70oC and let thaw on ice.
WARNING: When handling frozen competent cells, it is important to KEEP IT
COOL! Frozen cells are very sensitive to being warm and will not work as well if not
kept on ice as much as possible.
2. Place the cells on benchtop to thaw. Replace on ice.
3. After mixing gently, aliquot ~25 L of cells into chilled, 17x100 mm polypropylene
tube(s), e.g., Falcon 2059. Note: 25 L is enough for Transformation while 50 L is
required for Ligation.
4. Add 1.25 L or less plasmid DNA (the volume of DNA solution should not exceed
5% of the volume of the competent cells or no more than 50 ng in a volume of 10 L
or less). You don’t need very much plasmid to get transformed colonies. Generally
around 20 ng (nanograms) is enough.
5. Gently swirl tube(s) for a few seconds to mix by finger flicking tube. Note: If a
control is desired, repeat this step with 1-2 L (10-20 ng) of pUC19 in a separate
tube.
6. Then incubate tube on ice for 30 minutes.
7. Heat shock tube(s) by placing in 42oC water bath for ~45 seconds without shaking.
8. Replace tube(s) on ice for 2-5 minutes.
9. Add 900 L of S.O.C. (2% Tryptone, 0.5% Yeast Extract, 0.4% glucose, 10 mM
NaCl, 2.5 mM KCl, 10 mM MgCl2, & MgSO4).
10. Gently shake and incubate tube(s) at ~200 rpm for 30-60 minutes at 37oC.
11. Spread on LB agar plates containing appropriate antibiotic (e.g., 100 g/mL
ampicillin for control pUC19). Tip: Spread by evenly spreading cells over the plate
using a “hockey puck” spreader. Be sure that you have dipped your spreader in
ethanol and flamed it before spreading.
12. Place in the 37oC incubator with the agar side up and the lid side down. Incubate
overnight and remove to fridge (-4oC) sometime the next day.
Reagents/Solutions:
1. SOC Medium (1 Liter)
800 mL of deionized H2O
20 g Bacto-Tryptone
5 g Bacto-Yeast Extract
0.5 g NaCl
Shake until the solutes have dissolved. Add 10 mL of a 250 mM solution of KCl.
(This solution is made by dissolving 1.86 g of KCl in 100 mL of deionized H2O.)
adjust pH to 7.0 with 5 N NaOH (~0.2 mL). Adjust the volume of the solution to 1
Liter with deionized H2O. Autoclave.
Just before use, add 5 mL of a sterile solution of 2 M MgCl2. (This solution is made
by dissolving 19 g of MgCl2 in 90 mL of deionized H2O. Adjust the volume of the
solution to 100 mL with deionized H2O. Autoclave).
*20 mM glucose (After SOC has been autoclaved, allow to cool to 60oC or less and
then add 20 mL of a sterile 1 M solution glucose. This solution is made by dissolving
18 g of glucose in 90 mL of deionized H2O. after the sugar has dissolved, adjust
volume of the solution to 100 mL with deionized H2O and sterilize by filtration
through a 0.22-micron filter.)
2. 3 M NaOAc pH 5.2 (1 Liter)
If FW=82.03 g/mol, add:
246.1 g CH3COONa (anhydrous)
800 mL deionized H2O
pH to 5.2 with Glacial Acetic Acid
Bring to volume of 1 Liter
Autoclave
3. LB agar plates (1 Liter)
800 mL d H2O
10 g Bacto-Tryptone
5 g Bacto-Yeast extract
10 g NaCl
pH to 7.0
Bring to volume of 1 Liter
*Autoclave
*Just before autoclaving, add 15 g of Bacto-agar solidifying agent/1 Liter LB
Medium
Useful Tables:

Antibiotic Solutions
Ampicillin
Carbenicillin
Chloramphenicol
Kanamycin
Streptomycin
Tetracycline
Stock solution
concentrations
storage
50 mg/ml
in H20
-20oC
50 mg/ml
in H20
-20oC
34 mg/ml
in ethanol
-20oC
10 mg/ml
in H20
-20oC
10 mg/ml
in H20
-20oC
50 mg/ml
in ethanol
-20oC
a
Working concentration
Stringent
Relaxed
plasmids
plasmids
20 g/mL
60g/mL
20 g/mL
60g/mL
25 g/mL
170g/mL
10 g/mL
50g/mL
10 g/mL
50g/mL
10 g/mL
50g/mL
Stock solutions of antibiotics dissolved in H2O should be sterilized by filtration through a 0.22-micron filter.
Antibiotics dissolved in ethanol need not be sterilized. Store solution in light-tight containers.
b
Magnesium ions are antagonists of tetracycline. Use media without magnesium salts (e.g., LB Medium) for selection
of bacteria resistant to tetracycline.

Media Containing Agar or Agarose
Prepare liquid media according to the recipes given above. Just before autoclaving, add
one of the following:
Bacto-agar
(for plates) 15 g/Liter
Bacto-agar
(for top agar) 7 g/Liter
Agarose
(for plates) 15 g/Liter
Agarose
(for top agar) 7 g/Liter