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Cloning and Expression

S-Gal FAQs

S-Gal™/LB Agar Blend — Frequently Asked Questions
What purpose does the ferric ammonium citrate component serve in the S-Gal/LB Agar blend?
Ferric ions provided by the ferric ammonium citrate are chelated by the by-product of S-Gal, created when it is cleaved by b-galactosidase, to form the black precipitate.  Without it, the insoluble black precipitate would not form.

How do I prepare S-Gal/LB Agar Blend?
The 500 ml-packet of S-Gal/LB Agar Blend is mixed with deionized water and autoclaved for 20 minutes at 121°C and 15 psi. For preparation of microwaved medium, the mix is heated until boiling is first observed, followed with mixing, to allow the remaining insoluble agar component to go into solution. Heat for brief (15 to 30 seconds) intervals, if necessary, followed with continued mixing until blend is in solution. Antibiotics can be added post-autoclaving or -microwaving.

Will S-Gal/LB Agar Blend work with the same cloning vectors I'm currently using for blue-white color selection?
Yes. As with X-gal, S-Gal is cleaved by b-galactosidase, which is expressed in the absence of cloned DNA inserts. Black S-Gal-stained colonies or plaques indicate the absence of a cloned insert in the vector. White or cream-colored colonies or plaques denote the presence of vector with DNA insert.

Since color development is determined by b-galactosidase expression with both S-Gal and X-gal, is there any difference in the rate of color development of S-Gal versus X-gal-stained colonies or plaques?
Tests show that the rate of color development is essentially identical for S-Gal and X-gal.  However, the intense black stain of S-Gal stained colonies or plaques is more easily discerned at earlier time points throughout color development than with X-gal staining.

When cloning very small DNA fragments (less than 50 base pairs) that are 'in-frame' with the b-galactosidase gene, the results seen with X-gal are generally lighter blue colonies or plaques, or white colonies or plaques with "blue centers". Will this also be the case with S-Gal/LB Agar Blend?
Yes. A positive will be observed as a white or cream-colored colony or plaque with a black center.

Is S-Gal water-soluble?
S-Gal has been shown to be approximately 70% more water-soluble than X-gal.  Salt forms of  S-Gal, currently under development, are soluble at > 50 mg/ml in H20.

Is S-Gal light sensitive?
No. Test plates exposed to ambient laboratory light for two weeks performed as well as plates stored in the dark for two weeks.

Can prepared S-Gal/LB Agar Blend be re-melted for repeated use?
Yes. Autoclaved medium stored at 40C  was taken through six cycles of microwaving, with intervals of storage at 40C, without affecting performance of the medium.

Is there any negative effect on performance of S-Gal/LB Agar Blend caused by over microwaving?
Potentially.  When small volumes of medium, <50 ml, are microwaved, the temperature of the solution may exceed 100
0C (super-heating ). This can result in poor or no microbial growth due to degradation of the nutrients in the medium, not due to a breakdown of S-Gal . This phenomenon is less likely with large volumes, but the customer should be aware of this risk. Typically, heating to the point of boiling, followed with mixing, is adequate for dissolving the medium.

How long can autoclaved S-Gal/LB Agar Blend be held at 500C without affecting performance of the medium?
It is often convenient, and safer for handling, to place autoclaved or microwaved medium in a 50-520C water bath. Tests have shown that incubation at this temperature for up to one hour does not affect the performance of medium.

How stable is prepared S-Gal/LB Agar Blend?
Prepared medium stored  at 40C for two weeks showed no effective loss in performance.

Is S-Gal toxic?

When preparing medium by microwaving, should I be concerned about fungal or other microbial growth that would not be inhibited by added antibiotic?
Microwave heating is generally sufficient to remove most antibiotic-resistant micro-organisms.  In our hands, medium prepared by microwave was incubated at 370C for 60 hours without any detectable microbial growth. Microwaved medium stored for up to two weeks at 40C, then placed  at 370C for 48 hours, showed no microbial growth.

What bacterial strains can be used with S-Gal/LB Agar Blend?
Basically, any E. coli that is suitable for blue-white screening applications should work well with S-Gal/LB Agar Blend.  The following common strains have been shown to work well with S-Gal/LB Agar Blend.
Strain : Trademarked by :
JM 101
JM 109
DH 5 a Life Technologies, Inc.
DH 10 b Life Technologies, Inc.
XL1-Blue Stratagene, Inc.
XL10-Gold Stratagene, Inc.
TOP 10 Invitrogen, Inc.
TOP 10F1 Invitrogen, Inc.
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