Team:Imperial College London/Wetlab/Protocols/Restriction

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(New page: =Restriction Assay= Font: Calibri ===Solutions to be Prepared Beforehand=== * 5ml Lysis Buffer A. Method B – Mehling et. al (1995) a. 0.5‐1.0 g of cells are resuspended in 5mL lysi...)
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=Restriction Assay=
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{{Imperial/09/TemplateTop}}
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Font: Calibri
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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)
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=== Restriction Protocol ===
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a. 0.5‐1.0 g of cells are resuspended in 5mL lysis buffer (see below)
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<br>
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b. Incubate for 30‐80 min at 37C
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Perform for Dam +ve, -ve and TOP10 Strains
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c. Add 500uL of 5M NaCl solution
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In a PCR tube, mix up the following: <br>
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d. Agitate the suspension on a vortex mixer until the cell suspension
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* 5ul Genome DNA from Prep
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becomes translucent
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* 2ul of H2O
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e. Lyse cells by adding 1.2 mL of 10% SDS, mix thoroughly
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* 1ul of 10X BSA
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f. Incubate lysates for 15‐30 min at 65C
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* 1ul of 10X B2
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g. Add 2.4mL of 5M phosphate acetate
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* 0.5ul of TaqI
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h. Mix by vortexing
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* 0.5ul of DpnII
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i. Leave on ice for a minimum of 20 min (not much longer than)
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j. Remove precipitate by centrifugation for 30 min at 6000 rpm. Pour
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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
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least 40 min before adding the TE buffer.
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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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Latest revision as of 10:31, 30 September 2009



Restriction Protocol


Perform for Dam +ve, -ve and TOP10 Strains In a PCR tube, mix up the following:

  • 5ul Genome DNA from Prep
  • 2ul of H2O
  • 1ul of 10X BSA
  • 1ul of 10X B2
  • 0.5ul of TaqI
  • 0.5ul of DpnII
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