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| - | =Restriction Assay=
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| - | === Motivation ===
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| - | In order to investigate the effects of the restriction enzymes on the genetic material, we decided to perform a genomic prep, and to perform a restriction digest. Using methylated and unmethylated DNA from different strains,
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| | | | |
| - | === Strains Used ===
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| - | For comparison we used both Top10, our Dam positive chosen chassis, and a Dam negative contol, GM2163 (almost identical to ER2925). <br><br>
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| | | | |
| - | ===Solutions to be Prepared Beforehand=== | + | === Restriction Protocol === |
| - | * 5ml Lysis Buffer
| + | <br> |
| - | ** <i>25mM Tris
| + | Perform for Dam +ve, -ve and TOP10 Strains |
| - | ** 25mM EDTA (pH8.0)
| + | In a PCR tube, mix up the following: <br> |
| - | ** 10-15mg Lysosyme
| + | * 5ul Genome DNA from Prep |
| - | ** 50ug/ml RNaseA </i> <br>
| + | * 2ul of H2O |
| - | * 500ul of 5M NaCl Solution | + | * 1ul of 10X BSA |
| - | * 1.2ml of 10% SDS | + | * 1ul of 10X B2 |
| - | * 2.4ml of 5M Phosphate Acetate | + | * 0.5ul of TaqI |
| - | * 700ul of 50mM Tris/ 10mM EDTA | + | * 0.5ul of DpnII |
| - | * 75uL of 3M Sodium Acetate | + | |
| - | * 100uL of TE (10mM Tris/1mM EDTA, pH=8.0) | + | |
| - | | + | |
| - | === Genomic Prep Protocol ===
| + | |
| - | Method B – Mehling et. al (1995)<br>
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| - | a. 0.5‐1.0 g of cells are resuspended in 5mL lysis buffer (see below)<br>
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| - | b. Incubate for 30‐80 min at 37C<br>
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| - | c. Add 500uL of 5M NaCl solution<br>
| + | |
| - | d. Agitate the suspension on a vortex mixer until the cell suspension becomes translucent. <br>
| + | |
| - | e. Lyse cells by adding 1.2 mL of 10% SDS, mix thoroughly<br>
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| - | f. Incubate lysates for 15‐30 min at 65C<br>
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| - | g. Add 2.4mL of 5M phosphate acetate<br>
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| - | h. Mix by vortexing<br>
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| - | i. Leave on ice for a minimum of 20 min (not much longer than)<br>
| + | |
| - | j. Remove precipitate by centrifugation for 30 min at 6000 rpm. Pour supernatant into clean tube.<br>
| + | |
| - | k. Adjust volume of the supernatant to 8mL using isopropanol (about 1mL each)‐Recover DNA by precipitation<br>
| + | |
| - | l. Mix and incubate tubes at ‐20C for 30 min <br>
| + | |
| - | m. Pellet DNA at 20,000 x g for 15 min <br>
| + | |
| - | n. Pour off supernatant, let pellets dry for 10 min <br>
| + | |
| - | o. Dissolve precipitate in 700 uL of 50 mM Tris/10mM EDTA (pH 8.0)<br>
| + | |
| - | p. Transfer the solution to an eppendorf tube (1.5 mL microfuge tube), Spin off insoluble substances (10 min)<br>
| + | |
| - | q. Transfer aqueous phase to a 1.5mL microfuge tube<br>
| + | |
| - | r. Add 75uL 3M sodium acetate and 500uL isopropanol<br>
| + | |
| - | s. Mix Well and centrifuge solution for 30s to 2 min<br>
| + | |
| - | t. Wash pellet with cols 70% ethanol, dried and dissolved in 100uL TE(10mM Tris/1mM EDTA, pH=8.0), or distilled H2O – let pellet dry at least 40 min before adding the TE buffer.<br>
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