05/08/13

From 2013.igem.org

(Difference between revisions)
(Created page with "==Digestion of ligated plasmid==")
 
(11 intermediate revisions not shown)
Line 1: Line 1:
 +
<div style="font-family:arial;padding:5px;border-radius:5px;border:5px solid #FF2800;">
 +
{| style="color:#87EA00;background-color:#FFFFFF;" cellpadding="2" cellspacing="2" border="0" bordercolor="#000000" width="100%" align="center"
 +
!align="center"|[[Team:Leicester|Home]]
 +
!align="center"|[[Team:Leicester/Team|Team]]
 +
!align="center"|[https://igem.org/Team.cgi?year=2013&team_name=Leicester Official Team Profile]
 +
!align="center"|[[Team:Leicester/Project|Project]]
 +
!align="center"|[[Team:Leicester/Parts|Parts Submitted to the Registry]]
 +
!align="center"|[[Team:Leicester/Modeling|Modeling]]
 +
!align="center"|[[Team:Leicester/Notebook|Notebook]]
 +
!align="center"|[[Team:Leicester/Safety|Safety]]
 +
!align="center"|[[Team:Leicester/Attributions|Attributions]]
 +
|}
 +
</div>
 +
 +
 +
<p>
 +
 +
==Isolation of ligated plasmid==
 +
*The ligated plasmid- chloramphenicol backbone and limonene biobrick, were isolated from cells that were incubated overnight in broth by miniprep.
 +
* Protocols from both Bioline kit and Thermoscientific were used for the miniprep.
 +
* there were 6 tubes containing the plasmid.
 +
* The plasmid concentration in each of the 6 tubes were measured using nanodrop.
 +
* 200ng of plasmid from each of the 6 different tube was used for digestion.
 +
 +
{|border=1
 +
|sample||volume(ul)||concentration (ng/ul)||yield(ug)
 +
|-
 +
|5.1||99||75||7.4
 +
|-
 +
|5.2||100||44.2||4.4
 +
|-
 +
|5.3||98||46.7||4.6
 +
|-
 +
|10.1||46||61.8||2.84
 +
|-
 +
|10.2||44||77.1||3.39
 +
|-
 +
|10.3||47||13.7||0.64
 +
|}
 +
==Digestion of ligated plasmid==
==Digestion of ligated plasmid==
 +
*Master Mix (changes were made from the protocol)
 +
**2ul of NEB Buffer
 +
**1ul of EcoRI
 +
**1ul of PstI
 +
**varying concentration of dH2O and plasmid depending on overall concentration of plasmid in each tube.
 +
**Final volume of each of the 6 tubes was 20ul.
 +
 +
==Gel Electrophoresis==
 +
*The product of digestion was run on agarose gel
 +
**This was done in order to confirm the presence of the limonene biobrick
 +
[[File:iGem_Digested_050813.jpg]]
 +
*The lanes are in the following order:
 +
**Marker, Sample 5.1, Sample 5.2, Sample 5.3, Sample 10.1, Sample 10.2, Sample 10.3, Marker.
 +
*We were expecting to see a 2kb and 5kb band. These bands appeared for samples 5.1, 10.1,10.2 and 10.3. To further determine the composition of samples, we did a single digest on the samples.
 +
 +
==Single digest==
 +
*For samples 5.1, 10.1, 10.2 and 10.3 two single digests each were made with EcoRI and PstI, giving a total of 8 samples.
 +
*The samples are run on 0.8% agarose gel.

Latest revision as of 12:30, 6 August 2013

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


Contents

Isolation of ligated plasmid

  • The ligated plasmid- chloramphenicol backbone and limonene biobrick, were isolated from cells that were incubated overnight in broth by miniprep.
  • Protocols from both Bioline kit and Thermoscientific were used for the miniprep.
  • there were 6 tubes containing the plasmid.
  • The plasmid concentration in each of the 6 tubes were measured using nanodrop.
  • 200ng of plasmid from each of the 6 different tube was used for digestion.
samplevolume(ul)concentration (ng/ul)yield(ug)
5.199757.4
5.210044.24.4
5.39846.74.6
10.14661.82.84
10.24477.13.39
10.34713.70.64

Digestion of ligated plasmid

  • Master Mix (changes were made from the protocol)
    • 2ul of NEB Buffer
    • 1ul of EcoRI
    • 1ul of PstI
    • varying concentration of dH2O and plasmid depending on overall concentration of plasmid in each tube.
    • Final volume of each of the 6 tubes was 20ul.

Gel Electrophoresis

  • The product of digestion was run on agarose gel
    • This was done in order to confirm the presence of the limonene biobrick

IGem Digested 050813.jpg

  • The lanes are in the following order:
    • Marker, Sample 5.1, Sample 5.2, Sample 5.3, Sample 10.1, Sample 10.2, Sample 10.3, Marker.
  • We were expecting to see a 2kb and 5kb band. These bands appeared for samples 5.1, 10.1,10.2 and 10.3. To further determine the composition of samples, we did a single digest on the samples.

Single digest

  • For samples 5.1, 10.1, 10.2 and 10.3 two single digests each were made with EcoRI and PstI, giving a total of 8 samples.
  • The samples are run on 0.8% agarose gel.

Retrieved from "http://2013.igem.org/05/08/13"