Restriction Assay

Motivation

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,

Strains Used

For comparison we used both Top10, our Dam positive chosen chassis, and a Dam negative contol, GM2163 (almost identical to ER2925).

Solutions to be Prepared Beforehand

• 5ml Lysis Buffer
  • 25mM Tris
  • 25mM EDTA (pH8.0)
  • 10-15mg Lysosyme
  • 50ug/ml RNaseA
    
• 500ul of 5M NaCl Solution
• 1.2ml of 10% SDS
• 2.4ml of 5M Phosphate Acetate
• 700ul of 50mM Tris/ 10mM EDTA
• 75uL of 3M Sodium Acetate
• 100uL of TE (10mM Tris/1mM EDTA, pH=8.0)

Genomic Prep Protocol

Method B – Mehling et. al (1995)
a. 0.5‐1.0 g of cells are resuspended in 5mL lysis buffer (see below)
b. Incubate for 30‐80 min at 37C
c. Add 500uL of 5M NaCl solution
d. Agitate the suspension on a vortex mixer until the cell suspension becomes translucent.
e. Lyse cells by adding 1.2 mL of 10% SDS, mix thoroughly
f. Incubate lysates for 15‐30 min at 65C
g. Add 2.4mL of 5M phosphate acetate
h. Mix by vortexing
i. Leave on ice for a minimum of 20 min (not much longer than)
j. Remove precipitate by centrifugation for 30 min at 6000 rpm. Pour supernatant into clean tube.
k. Adjust volume of the supernatant to 8mL using isopropanol (about 1mL each)‐Recover DNA by precipitation
l. Mix and incubate tubes at ‐20C for 30 min
m. Pellet DNA at 20,000 x g for 15 min
n. Pour off supernatant, let pellets dry for 10 min
o. Dissolve precipitate in 700 uL of 50 mM Tris/10mM EDTA (pH 8.0)
p. Transfer the solution to an eppendorf tube (1.5 mL microfuge tube), Spin off insoluble substances (10 min)
q. Transfer aqueous phase to a 1.5mL microfuge tube
r. Add 75uL 3M sodium acetate and 500uL isopropanol
s. Mix Well and centrifuge solution for 30s to 2 min
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.

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