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| - | =Restriction Assay=
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| - | ===Solutions to be Prepared Beforehand===
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| - | * 5ml Lysis Buffer
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| - | A. Method B – Mehling et. al (1995)
| + | |
| - | a. 0.5‐1.0 g of cells are resuspended in 5mL lysis buffer (see below)
| + | === Restriction Protocol === |
| - | b. Incubate for 30‐80 min at 37C
| + | <br> |
| - | c. Add 500uL of 5M NaCl solution
| + | Perform for Dam +ve, -ve and TOP10 Strains |
| - | d. Agitate the suspension on a vortex mixer until the cell suspension
| + | In a PCR tube, mix up the following: <br> |
| - | becomes translucent
| + | * 5ul Genome DNA from Prep |
| - | e. Lyse cells by adding 1.2 mL of 10% SDS, mix thoroughly
| + | * 2ul of H2O |
| - | f. Incubate lysates for 15‐30 min at 65C
| + | * 1ul of 10X BSA |
| - | g. Add 2.4mL of 5M phosphate acetate
| + | * 1ul of 10X B2 |
| - | h. Mix by vortexing
| + | * 0.5ul of TaqI |
| - | i. Leave on ice for a minimum of 20 min (not much longer than)
| + | * 0.5ul of DpnII |
| - | j. Remove precipitate by centrifugation for 30 min at 6000 rpm. Pour
| + | |
| - | supernatant into clean tube.
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| - | k. Adjust volume of the supernatant to 8mL using isopropanol (about 1
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| - | mL each)‐Recover DNA by precipitation
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| - | l. Mix and incubate tubes at ‐20C for 30 min
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| - | m. Pellet DNA at 20,000 x g for 15 min
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| - | n. Pour off supernatant, let pellets dry for 10 min
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| - | o. Dissolve precipitate in 700 uL of 50 mM Tris/10mM EDTA (pH 8.0)
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| - | p. Transfer the solution to an eppendorf tube (1.5 mL microfuge tube),
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| - | Spin off insoluble substances (10 min)
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| - | q. Transfer aqueous phase to a 1.5mL microfuge tube
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| - | r. Add 75uL 3M sodium acetate and 500uL isopropanol
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| - | s. Mix Well and centrifuge solution for 30s to 2 min
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| - | t. Wash pellet with cols 70% ethanol, dried and dissolved in 100uL TE
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| - | (10mM Tris/1mM EDTA, pH=8.0), or distilled H2O – let pellet dry at
| + | |
| - | least 40 min before adding the TE buffer.
| + | |
| - | B. Lysis Buffer
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| - | a. 25mM Tris
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| - | b. 25mM EDTA, pH 8.0
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| - | c. 10‐15 mg Lysosyme
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| - | d. 50ug/mL RNaseA
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