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Plates for Bacterial Growth
LB Plates: Selection by Antibiotic
LB Agar plates with 100 µg/mL Ampicillin. 100mm, 20 plates, Sterile Cat.No. L1004
Our Price: $31.84
Sale Price: $
: LB Agar plates with 100µg/mL Ampicillin. 100mm, 20 plates. Sterile.
: For use with recombinant strains of E.coli containing plasmids with the resistance gene to Ampicillin (
bla or amp
) such as pBluescript, pGEM, or the pUC series of plasmids.
1.0% Tryptone (10.0g/L)
0.5% Yeast Extract (5.0g/L)
1.0% Sodium chloride (NaCl) (10.0g/L)
1.5% Agar (15.0g/L)
Store at 2-8
Certificate of Analysis available upon request. To request
CAS: 69-53-4 (Ampicillin Sodium salt: CAS: 69-52-3)
Molecular Weight: 349.41g/mol (Ampicillin Sodium salt: 371.39g/mol)
S (Ampicillin Sodium salt: C
Stock Solutions: Ampicillin is made at 100mg/ml in water (or 100% Ethanol); store aliquots at -20
Final Concentration: Ampicillin is used at 50µg/ml for low copy plasmids and at 100µg/ml for high-copy plasmids.
Mode of Action: Bactericidal. Ampicillin only kills growing
. Ampicillin inhibits cell wall synthesis by inhibiting formation of the peptidoglycan cross-linkages.
Mode of Resistance: Secreted β−lactamase in bacteria hydrolyzes ampicillin before it enters the cell. In plasmids, resistance conferred by the product of the TEM-1 β−lactamase (
) gene from Tn3.
Ampicillin is used as a selectable marker in many commonly used plasmids. A functional Ampicillin gene will confer resistance to cells containing the plasmid. Cultures of
are grown on media with Ampicillin and only those cells transformed with plasmids containing the Ampicillin resistance gene (known as
) will survive.
Commonly used plasmids in cloning using
for selection include:
1. pUC19: a very high copy vector resulting in several hundred copies of plasmid per cell, utilizing a modified version of pMB1 as an origin of replication
2. pBR322: a high copy vector resulting in approximately 20 copies of plasmid per cell, utilizing colE1 as an origin of replication
3. pSC101: a low copy vector resulting in 5 copies of plasmid per cell, utilizing pSC101 as an origin of replication
Ampicillin is a β-lactam antibiotic, a member of the aminopenicillin family and is considered equivalent to its successor, Amoxicillin in terms of spectrum and level of activity. Ampicillin has been used widely to treat bacterial infections since 1961. Until the introduction of ampicillin, penicillin therapies were effective only against Gram-positive organisms such as
. Ampicillin also is known to be active against Gram negative organisms such as
The presence of an amino group on Ampicillin distinguishes its structure from Penicillin and this amino group helps the drug penetrate the outer membrane of Gram negative bacteria. The rigid cell wall of susceptible organisms is due in part to a thin layer of cross-linked peptidoglycan, one to two molecules thick. The long glycan chains are composed entirely of amino sugars and are cross-linked by peptide chains. The penicillins inhibit the final stage of synthesis of cross-links, which occurs outside the cell and is catalyzed by a transpeptidase enzyme. Ampicillin acts as a competitive inhibitor of transpeptidase.
Ampicillin inhibits bacterial cell wall synthesis by binding to one or more of the penicillin binding proteins (PBPs) that inhibit the final transpeptidation step of peptidoglycan synthesis in bacterial cell walls, thus inhibiting cell wall biosynthesis. Members of the penicillin family are most active against bacteria in the logarithmic phase of growth and have relatively little effect in the stationary phase, when synthesis of peptidoglycan is suppressed. Bacteria eventually lyse due to ongoing activity of cell wall autolytic enzymes (autolysins and murein hydrolases) and cell wall assembly is arrested. Penicillins also inhibit enzymes necessary for the rod-like structure of
and septum formation during division.
The enzyme responsible for degrading ampicillin is β-lactamase, in reference to the β-lactam structure of ampicillin and related drugs. β-lactamase is present in small amounts in the periplasmic space of gram-negative bacteria. β-lactamase catalyzes hydrolysis of the cyclic amide bond of β-lactam rings, with concomitant detoxification of ampicillin. β-lactamases hydrolyze the ampicillin in the surrounding medium of bacteria and create a protected zone in which antibiotic-sensitive colonies can grow. This leads to the appearance of non-transformed satellite colonies.
The appearance of satellite colonies can be ameliorated by using Carbenicillin (instead of ampicillin) in the selective media. Carbenicillin, due to its chemical structure, is more resistant to hydrolysis by β-lactamases. The downside is cost. Carbenicillin is much more expensive than Ampicillin. Sometimes mixtures of Carbenicillin and Ampicillin are used.
It is important to stress that since Αmpicillin inhibits peptidoglycan cross-linking, which occurs during growth, the antibiotic is active only in growing cultures and is most active in the log phase of growth.
Studier (1990) found that secreted β-lactamase will quickly consume all the ampicillin in culture. A culture of
in the stationary phase of growth contains such a high concentration of secreted β-lactamase that a 1:200-1:1000 dilution of this culture will still be able to consume all the fresh ampicillin before all non-resistant cells (in stationary phase) have been killed. The authors recommend not allowing cultures to reach stationary phase to obtain a high proportion of recombinant cells (containing plasmid).
1. Green and Sambrook, Molecular Cloning, A Laboratory Manual, 4th Edition, (2012)
2. Ausubel et al; Short Protocols in Molecular Biology (2002)
3. Sambrook and Russell, Molecular Cloning, A Laboratory Manual, 3rd Edition (2001)
4. Evans, Galindo, Olarte and Falkow. Beta-lactamase of R factors. J. Bacteriol. 96(4): 1441-1442 (1968).
5. Anderson and Datta. Resistance to Penicillins and its transfer in Enterobacteriaceae. Lancet 1(7382) 407-409 (1965).
6. Datta and Richmond. The purification and properties of a penicillinase whose synthesis is mediated by an R-factor in Escherichia coli. J. Biochem. 98(1) 204-209 (1966).
7. Studier, Rosenberg, Dunn and Dubendorff. Use of T7 RNA polymerase to direct expression of clones genes. Methods Enzymol. 185:60-89 (1990).
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