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From 2013.igem.org

Contents 1 Isolating plasmid 2 Digesting the plasmids for restriction mapping 3 Running an agarose gel 4 Glycerol stocks 5 Sonicating Herring sperm DNA

Isolating plasmid

• From overnight culture, took 3ml of culture and centrifuged into pellet of samples 5.1, 10.1, 10.2, 10.3
• Isolated the plasmid using omega bio-tek Plasmid mini kit I

• Concentrations from nano drop are shown in the table below

Sample	Volume(ul)	Conc.(ng/ul)	260/280	260/230
5.1	92	65.8	1.87	1.75
10.1	88	36.9	1.80	1.77
10.2	89	48.3	1.79	1.98
10.3	94	17.4	1.84	1.59

Digesting the plasmids for restriction mapping

• Digesting 200ng of DNA with SacI
  • 1ul of SacI
  • 2ul of NEB buffer 1.1
  • DNA volumes added for samples 5.1 to 10.3:
    • 3ul; 5ul; 4ul; 11.5ul
  • Water added for samples 5.1 to 10.3:
    • 14ul; 12ul; 13ul; 5.5ul
• Digestion in 37C water bath for 30mins
• Heat kill at 80C for 20mins

Running an agarose gel

• Add 5ul of 6x orange G to each sample
• 1kb marker is used
• Wells are loaded in the following order:
  • 5ul of marker, 25ul of samples 5.1; 10.1; 10.2; 10.3, 5ul of marker

• The lanes are as expected.
• Samples 5.1, 10.1 and 10.3 were expected to show 3 fragments, sized 2070, 1890 and 1529bp. This is what is shown on the gel.
• Sample 10.3 only has one SacI restriction site and when cut, a band of 2070bp is expected. That is also shown on the gel.

Glycerol stocks

• Took 750ul from overnight culture and centrifuged
• Supernatant was removed
• Pellet resuspended in 375ul of HMFM
• Samples were then frozen at -80

Sonicating Herring sperm DNA

• The herring Sperm was to viscous, so to reduce viscosity the sonicator was used
  • 1ml of core DNA used in 2 tubes, 1 control and 1 to go in the sonicator
  • The control tube was left on the bench at room temperature
  • The 2nd tube was put in the sonicator for the folowing times: 2mins, 2mins, 5mins, 5mins, 20mins, 20mins, 30mins
  • Each time both tubes were filmed to compare viscosity