SSC buffer dùng để làm gì?


SSC buffer là gì:

In biochemistry and molecular biology, the saline-sodium citrate (SSC) buffer is used as a hybridization buffer, to control stringency for washing steps in protocols for Southern blotting, in situ hybridization, DNA Microarray or Northern blotting. 20X SSC may be used to prevent drying of agarose gels during a vacuum transfer.

A 20X stock solution consists of 3 M sodium chloride and 300 mM trisodium citrate (adjusted to pH 7.0 with HCl).

Có 2 ứng dụng của SSC buffer

This buffer is used for the denaturation of DNA for the screening of DNA libraries (e.g. genomic, cDNA or YAC library). The probe for screening the library will only bind to the immobilized DNA of the library, if this DNA is in a single stranded state. The commonly used working concentration is 2X SSC (Ref. 4, e.g. p. 6.1.3).

The second major application is the use of SSC as transfer buffer for the blotting of DNA after agarose or polyacrylamide gel electrophoresis onto nitrocellulose or nylon membranes (Southern-Blot). In case of using nitrocellulose membranes, a high SSC concentration (20X; high ionic strength) should be employed, since smaller DNA fragments might be lost at concentrations of 10X SSC or below. A good result for nylon membranes can be expected using 20X and 10X SSC. Depending on the brand of the nylon membrane other conditions may result in better hybridization rates (e. g. positive charged membranes with an alkaline transfer buffer).
If used as a transfer buffer, it is not necessary to filter SSC, but when used as in hybridization solutions, it is essential to filter SSC before use. SSC buffer from AppliChem will be filtered principally. SSC buffer may be replaced with the same concentration of SSPE buffer (A1397) in all experiments. SSPE has a greater buffering capacity. The buffering power of SSC can be increased by adding 0.3 % (w/v) sodium pyrophosphate.
In references 3 and 4 several techniques are described in detail:

* Other Ref:

One solution can be used for both prehybridization and hybridization and can be as simple as 5X SSC (diluted from 20X SSC: 0.3 M Na citrate, pH 7, 3 M NaCl), 1.0% protein blocker (either casein or BSA), 0.1% N-lauroylsarcosine, 0.02% sodium dodecyl sulfate.  This solution can be aliquoted and stored at -20°C or colder.

* Need to store at -20 degree or colder to keep BSA (protein nature) stable.

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