09/08/13

From 2013.igem.org

(Difference between revisions)
(→Sonicating Herring sperm DNA)
(→Sonicating Herring sperm DNA)
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**1ml of core DNA used in 2 tubes, 1 control and 1 to go in the sonicator
**1ml of core DNA used in 2 tubes, 1 control and 1 to go in the sonicator
**The 2nd tube was put in the sonicator for the folowing times: 2mins, 2mins, 5mins, 5mins, 20mins
**The 2nd tube was put in the sonicator for the folowing times: 2mins, 2mins, 5mins, 5mins, 20mins
-
**Each time the tubes were filmed to show viscosity
+
**Each time both tubes were filmed to compare viscosity

Revision as of 15:20, 9 August 2013

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Contents

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
SampleVolume(ul)Conc.(ng/ul)260/280260/230
5.19265.81.871.75
10.18836.91.801.77
10.28948.31.791.98
10.39417.41.841.59

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

Glycerol stocks

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

Restriction mapping

  • using isolated plasmid samples 5.1, 10.1, 10.2, 10.3

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 2nd tube was put in the sonicator for the folowing times: 2mins, 2mins, 5mins, 5mins, 20mins
    • Each time both tubes were filmed to compare viscosity

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