09/08/13

From 2013.igem.org

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==Digesting the plasmids for restriction mapping==
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*Digesting 200ng of DNA with SacI
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**1ul of SacI
 +
**2ul of NEB buffer 1.1
 +
**DNA volumes added for samples 5.1 to 10.3:
 +
***3ul; 5ul; 4ul; 11.5ul
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**Water added for samples 5.1 to 10.3:
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***14ul; 12ul; 13ul; 5.5ul
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*Digestion in 37C water bath for 30mins
 +
*Heat kill at 80C for 20mins
 +
 +
==Running an agarose gel==
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*Add 5ul of 6x orange G to each sample
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*1kb marker is used
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*Wells are loaded in the following order:
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**5ul of marker, 25ul of samples 5.1; 10.1; 10.2; 10.3, 5ul of marker
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[[File:lim_chlorodig_sacI_090813.jpg]]
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*The lanes are as expected.
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*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==
==Glycerol stocks==
-
*Took 750ul from overnight stock and centrifuged
+
*Took 750ul from overnight culture and centrifuged at full speed for ~2 minutes
*Supernatant was removed
*Supernatant was removed
*Pellet resuspended in 375ul of HMFM
*Pellet resuspended in 375ul of HMFM
-
*samples were then frozen at -80
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*Samples were then frozen at -80
-
==Restriction mapping==
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==Sonicating Herring sperm DNA==
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*using isolated plasmid samples 5.1, 10.1, 10.2, 10.3
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*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
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**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

Latest revision as of 15:12, 12 September 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
  • 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

Lim chlorodig sacI 090813.jpg

  • 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 at full speed for ~2 minutes
  • 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

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