CulGenex™

Antibiotic and Chromogenic Supplements

Cat. no. CG16 Ampicillin Solution 50mg/ml, 1.5ml amber tube, 1ml 6 tubes/box
Cat. no. CG21 Carbenicillin Solution 100mg/ml, 1.5ml amber tube, 0.5ml 6 tubes/box
Cat. no. CG20 Kanamycin Solution 50mg/ml, 1.5ml amber tube, 0.5ml 6 tubes/box
Cat. no. CG18 IPTG Solution 0.1M, 1.5ml amber tube, 1ml 6 tubes/box
Cat. no. CG17 X-GAL Solution 40mg/ml, 1.5ml amber tube, 1ml 6 tubes/box
Cat. no. CG19 X-GAL 40mg/ml and IPTG 0.1M Solution, 1.5ml amber tube, 1ml 6 tubes/box

INTENDED USE

Hardy Diagnostics CulGenex™ Antibiotic and Chromogenic Supplements are recommended for use in the selective and/or differential growth of recombinant strains of Escherichia coli used for genetic research. Antibiotics such as ampicillin, kanamycin and carbenicillin are typically used as selective agents to isolate strains that have taken up genes (e.g. plasmids) coupled to a gene encoding for resistance. X-GAL, used in conjunction with IPTG, is intended for the colorimetric detection of beta-galactosidase activity in recombinant strains.

This product is not intended to be used for the diagnosis of human disease.

SUMMARY

Ampicillin is widely used in molecular research and is effective against gram-negative and gram-positive microorganisms. This antibiotic inhibits cell-wall synthesis by interfering with peptidoglycan cross-linking. Bacterial resistance to ampicillin occurs through the bla gene, which encodes for the enzyme beta-lactamase. Beta-lactamase cleaves the beta-lactam bond of the antibiotic, essentially making it ineffective against the organism. Ampicillin is recommended for use with all pUC derived plasmids carrying the beta-lactamase gene (e.g. pUC19, pBluescript, pGEM, etc.) and is commonly used at a working concentration of 20ug/ml for stringent plasmids and a working concentration of 50ug/ml for relaxed plasmids. (6) In addition, ampicillin is traditionally added to all broth and agar media used for growing YAC (yeast artificial chromosome) libraries.

Carbenicillin is an ampicillin analog that binds and inhibits enzymes involved in the synthesis of bacterial cell walls. It is most active against isolates of Pseudomonas aeruginosa and certain indole-positive Proteus spp. normally resistant to ampicillin. The gene conferring resistance to ampicillin and its analogs, amp r , codes for beta-lactamase; carbenicillin is less sensitive to beta-lactamase than ampicillin. In addition, carbenicillin has a superior stability at low pH when compared to ampicillin, and research shows that the use of effective concentrations from 50 to 100ug/ml help prevent overgrowth of satellite colonies.

Kanamycin is effective against gram-negative and gram-positive bacteria and Mycoplasma spp. and binds to the 70S ribosomal subunit, eliciting chromosomal miscoding and resulting in dysfunctional translation of mRNA. Resistance to kanamycin is the result of the enzyme aminoglycoside phosphotransferase which modifies the antibiotic and prevents its interaction with ribosomes. Kanamycin is recommended for use in cell culture media at a concentration of 100ug/ml and is traditionally added to all broth and agar media used for growing PAC (P1-derived artificial chromosome) and cFugu ( Fugu rubripes ) libraries in Escherichia coli ; it is commonly used at a working concentration of 25ug/ml for PAC libraries and a working concentration of 30ug/ml for cFugu libraries. (3)

IPTG (isopropyl-beta-D-thiogalactopyranoside) is a non-metabolized analog of galactose used to induce expression of genes under control of the lac operon in Escherichia coli . It is used in conjunction with X-GAL to screen for "blue-white" colonies using vectors that support alpha complementation of beta-galactosidase to detect recombinant plasmids and phage.

X-GAL (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) is frequently used in "blue-white" colony screens to detect bacterial or phage clones containing the lac Z or lac Z-peptide gene; this additive turns blue when incubated in the presence of the enzyme beta-glactosidase. The gene encoding for beta-galactosidase is on several cloning plasmid standards (especially the pUC series and IGT11 vectors). When an inserted portion of DNA is placed in the correct restriction site, the lacZ gene is interrupted and the desired colony fails to turn blue.

Hardy Diagnostics CulGenex™ Antibiotic and Chromogenic Supplements are pre-mixed and ready to use. They are available by the individual vial.

FORMULA

Ingredients per milliliter of molecular grade water:*

Ampicillin Solution 50mg/ml (Cat. no. CG16):
Ampicillin 50.0mg

Carbenicillin Solution 100mg/ml (Cat. no. CG21):
Carbenicillin Disodium Salt 100.0mg

Kanamycin Solution 50mg/ml (Cat. no. CG20):
Kanamycin Disulfate Salt 50.0mg

IPTG Solution 0.1M (Cat. no. CG18):
IPTG 24.0mg

X-GAL Solution 40mg/ml (Cat. no. CG17):
X-GAL 40.0mg

X-GAL 40mg/ml and IPTG 0.1M Solution (Cat. no. CG19):
X-GAL 40.0mg
IPTG 24.0mg

* Adjusted and/or supplemented as required to meet performance criteria.

STORAGE AND SHELF LIFE

Storage: Upon receipt store chromogenic supplements (Cat. no. CG17, CG18 and CG19) at 2-8ºC. and antibiotic supplements (Cat. no. CG16, CG20 and CG21) < -20ºC. away from direct light. Products should not be used if there are any signs of deterioration, discoloration, contamination, or if the expiration date has passed. Products are light and temperature sensitive; protect from light, excessive heat, and moisture. Avoid excessive freezing and thawing of frozen supplements.


PRECAUTIONS


PROCEDURE

Consult listed references for information on specific protocols. (1,2,4-6)

Adding supplements to freshly made plates prior to pouring:

1. After autoclaving, allow the medium to cool to 50ºC. prior to incorporating supplements.

For adding chromogenic supplements:

2a. Add IPTG to a final concentration of 0.1mM IPTG (1ul IPTG stock solution per ml of media) and X-GAL to a final concentration of 40ug/ml (1ul of X-GAL stock solution per ml of media).

Note: X-GAL is commonly used at a final concentration of 40 to 50ug/ml in plated media and overlays. IPTG is commonly used at a final concentration of approximately 0.5mM in plated media and overlays, but its useful concentration varies from 0.1 to 2mM, depending upon the application. IPTG is also used in the over expression of proteins at a recommended concentration of 0.1 to 0.4mM, but the concentration will vary slightly with the vector and strain used.

For adding antibiotic supplements:

2b. If desired, add the appropriate selective agent(s) to the medium.

3. Aseptically, pour desired volume into sterile petri plates. Alternatively, aseptically spread 40 to 100ul of a stock solution of X-GAL/IPTG onto the solid agar surface of pre-poured plates and allow sufficient time for the solution to dry prior to inoculating the medium.

QUALITY CONTROL

Hardy Diagnostics tests CulGenex™ Ampicillin Solution 50mg/ml, Carbenicillin Solution 100mg/ml, Kanamycin Solution 50mg/ml, IPTG Solution 0.1M, X-GAL Solution 40mg/ml, and X-GAL 40mg/ml and IPTG 0.1M Solution for sterility and fill volume.

USER QUALITY CONTROL


Physical Appearance

CulGenex™ Ampicillin Solution 50mg/ml, Carbenicillin Solution 100mg/ml, Kanamycin Solution 50mg/ml, IPTG Solution 0.1M, X-GAL Solution 40mg/ml, and X-GAL 40mg/ml and IPTG 0.1M Solution should appear clear, and colorless.

REFERENCES

1. Ausubel, F.M., R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, K. Struhl, Editors. 2010. Current Protocols in Molecular Biology . John Wiley and Sons, Inc. Malden, MA.

2. Cseke, L.J., P.B. Kaufman, G.K. Podila, and C.J. Tsai. 2004. Handbook of Molecular and Cellular Methods in Biology and Medicine . CRC Press.Taylor & Francis LLC. Boca Raton, FL.

3. Pestka, S. 1975. The Use of Inhibitors in Studies on Protein Synthesis. Meth. in Enzymology ; 30:261-282.

4. Sambrook and Russell. 2001. Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press. Woodbury, New York.

5. Walker, J.M. 1984. Methods in Molecular Biology . The Humana Press Inc. Clifton, NJ.

6. Williams, S., B. Slatko, and J. McCarrey. 2006. Laboratory Investigations in Molecular Biology . Jones and Bartlett, Sudbury, MA.


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