09/08/13

From 2013.igem.org

(Difference between revisions)
(Glycerol stocks)
 
(18 intermediate revisions not shown)
Line 19: Line 19:
*Isolated the plasmid using omega bio-tek Plasmid mini kit I
*Isolated the plasmid using omega bio-tek Plasmid mini kit I
-
*Concentrations are shown in the table below
+
*Concentrations from nano drop are shown in the table below
{|border=1
{|border=1
|Sample||Volume(ul)||Conc.(ng/ul)||260/280||260/230
|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
 +
 
 +
[[File: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

Latest revision as of 15:12, 12 September 2013

Home Team Official Team Profile Project Parts Submitted to the Registry Modeling Notebook Safety Attributions

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