Now that you know how to prepare a restriction map, you decide to actually prepa
ID: 52149 • Letter: N
Question
Now that you know how to prepare a restriction map, you decide to actually prepare a digest reaction using some new restriction enzymes. First you need to plan your experiment and determine the exact protocol to follow (ie what ‘ingredients’ will go into your digestion reaction, how much of those ingredients, incubation conditions, length of incubation, etc). a) Describe the reaction mixture (including the volumes of each reagent you will need to add) by filling in the following table and then b) describe experimental conditions (ie temperature and time) that you will use to digest 1ug of plasmid DNA (Ci=100ng/uL) with 10U each of SpeI, and Xba I (New England Biolabs, Ci=20,000U/mL) in a total volume of 30uL. If needed for your reaction, the BSA being provided has an initial concentration of 100X and the NEB Buffers being provided have an initial concentration of 10X.
Hint: 20,000U/mL = 20U per microliter (ul).
Fill in the rest of the table – use 30uL total reaction volume
Experimental conditions for digestion (time, temperature):
Explanation / Answer
A)Reaction mixture :
pH: Most restriction enzymes are used between pH 7.2 and pH 8.5 as measured at the temperature of incubation. pH values outside of the optimal range may lead to star activity.
Mg2+: Commercially available restriction enzymes require Mg2+ as the only cofactor. Restriction enzyme activities are relatively insensitive to the Mg2+ concentration; similar rates are observed from 5-30mM. The presence of other divalent metal ions, especially Mn2+, may lead to star activity.
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. A few enzymes prefer acetate to chloride anions. Suboptimal ionic strength or type of ion may lead to star activity.
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. The Acetylated BSA provided with Promega's restriction enzymes has been modified and extensively tested to ensure that no degrading activities are present.
Glycerol: Glycerol is added to restriction enzyme storage buffers to prevent freezing at -20°C. Repeated freeze/thawing of restriction enzymes can reduce their activity. Some restriction enzymes show reduced specificity, or increased star activity, when the glycerol concentration in the final reaction is higher than 5% although many have normal specificity at glycerol concentrations as high as 10%.
Incubation Temperature: Most restriction enzymes show maximum activity at 37°C. A few enzymes require higher or lower temperatures for optimal activity (e.g., Taq I, 65°C; Sma I, 25°C). For incubations greater than 1 hour with high temperature enzymes, cover the reactions with a drop of mineral oil to prevent evaporation. Generally, the incubation temperature for the enzyme reflects the growth temperature of the bacterial strain from which it is derived. For enzymes that have temperature optima other than 37°C, Promega provides information on percent activity at 37°C on the Product Information sheet that is packaged with each enzyme. This type of information is particularly useful when performing double digests.
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 must take into account final ionic strength and must result in glycerol concentrations no higher than 5-10% in order to avoid star activity. Reaction volumes of 10-50µl per microgram of DNA are recommended.
B)experimental conditions:
components volumes
Sterile, nuclease-free water 23.7µl
Restriction enzyme 10X buffer 3µl(1X-Should be your working range)
BSA Acetylated (100X) 0.3µl(1X-Should be your working range)
DNA sample 0.2-1µg (preferable conc.) 1µl
Restriction enzyme,speI, 10U 1µl(optimal concentration is enough to prevent star activity)
Restriction enzyme,XbaI,10U 1µl(optimal concentration is enough to prevent star activity)
Final volume 30µl
Mix gently by pipetting. Centrifuge briefly at 12,000 x g in a micro centrifuge to collect the contents at the bottom of the tube.
Incubate at the 37oC temperature for 1-4 hours.
Addtional information:
DNA Restriction Enzyme: XbaI
T | CTAG A A GATC | TRelated Questions
Navigate
Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.