List and describe the purpose of each component of the restriction enzyme digest

Explanation:

Restriction enzymes are enzymes isolated from bacteria that recognize specific sequences in DNA and then cut the DNA to produce fragments, called restriction fragments

Restriction enzymes play a very important role in the construction of recombinant DNA molecules, as is done in gene cloning experiment

Components of the restriction enzyme digest include:

1) pH: Most restriction enzymes are used between pH 7.2 and pH 8.5 as measured at the temperature of incubation

2) Mg2+: Commercially available restriction enzymes require Mg2+ as the only cofactor whereas restriction enzyme activities are relatively insensitive to the Mg2+ concentration

3) Salt Concentration: Restriction enzymes are diverse in their response to ionic strength

Most are stimulated by 50-150mM NaCl or KCl while others are inhibited by salt concentrations higher than 20mM

4) BSA: Bovine Serum Albumin is used in restriction enzyme storage buffers and is added to digestion reactions to stabilize the enzyme

BSA can protect restriction enzymes from proteases, non- specific adsorption and harmful environmental factors such as heat, surface tension and interfering substances

Typically, the addition of 0.1mg/ml BSA will result in a 1.5 to 6-fold enhancement of enzyme activity

5) Glycerol: Glycerol is added to restriction enzyme storage buffers to prevent freezing at -20°C

Repeated freeze or thawing of restriction enzymes can reduce their activity and some restriction enzymes show reduced specificity when the glycerol concentration in the final reaction is higher than 5% although many have normal specificity at glycerol concentrations as high as 10%

6) Incubation Temperature: Most restriction enzymes show maximum activity at 37°C

Generally, the incubation temperature for the enzyme reflects the growth temperature of the bacterial strain from which it is derived

7) Volume: Viscous DNA solutions inhibit enzyme diffusion and can reduce enzyme activity

DNA concentrations that are too dilute can fall below the Km of the restriction enzyme and also affect enzyme activity,volume considerations is taken into account

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