09/09/13

From 2013.igem.org

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==Preparation of overnight broth==
 
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* Following on from [[06/09/13]] result,overnight broth was prepared yesterday from the single colonies that had grown on each of the 2 plates shown on [[06/09/13]].
 
-
 
+
==Miniprep of overnight broth==
-
 
+
* Following on from [[06/09/13]] result,overnight broth was prepared yesterday from the single colonies that had grown on each of the 2 plates shown on [[06/09/13]].
-
==Miniprep of broth==
+
* The broth, which contains cells that have been transformed with pSB1C3/TOD genes, was minipreped using the Omega Bio-Tek kit.
* The broth, which contains cells that have been transformed with pSB1C3/TOD genes, was minipreped using the Omega Bio-Tek kit.
* This would enable us to confirm which cells have the recircularised plasmid and those with the ligated product.
* This would enable us to confirm which cells have the recircularised plasmid and those with the ligated product.
==Measurement of DNA concentration==
==Measurement of DNA concentration==
 +
{| border=1
 +
|Samples ||Volume (ul)||Conc (ng/ul)||280/260||260/280
 +
|-
 +
|TOD X1||31.6||86.5||1.87||1.92
 +
|-
 +
|TOD X2||31.7||58.7||1.89||1.26
 +
|-
 +
|TOD X3||32.7||56.6||1.91||1.88
 +
|-
 +
|TOD X4||31.6||55.8||1.88||1.65
 +
|-
 +
|TOD X5||31.7||44.4||1.87||1.78
 +
|-
 +
|TOD X6||31.7||70.5||1.88||1.88
 +
|-
 +
|
 +
|-
 +
|TOD F1||32.4||39.6||1.91||1.59
 +
|-
 +
|TOD F2||33.7||49.4||1.91||1.65
 +
|-
 +
|TOD F3||32.3||47.4||1.89||1.71
 +
|-
 +
|TOD F4||31.6||43.7||1.94||1.73
 +
|-
 +
|TOD F5||36.5||57||1.89||1.50
 +
|-
 +
|TOD F6||38||59.4||1.89||1.61
 +
|-
 +
|
 +
|-
 +
|TOB G1||36.8||72.5||1.89||1.51
 +
|-
 +
|TOB G2||44.7||1.90||1.90||1.65
 +
|-
 +
|TOB G3||33.7||50.2||1.90||1.46
 +
|-
 +
|TOB G4||41.9||55||1.93||1.35
 +
|-
 +
|TOB G5||34||43||1.96||1.54
 +
|-
 +
|TOB G6||35||48||1.89||1.72
 +
 +
|}
 +
 +
DNA concentration was measured using Nanodrop.
 +
 +
==Double Digestion of pSB1C3/TOD genes==
 +
*The pSB1C3/TOD genes were double digested with (XbaI and SpeI) & (EcoRI and PstI)
 +
*The master mix for the (XbaI and SpeI) was;
 +
60ul of cut smart buffer
 +
5ul of XbaI
 +
10ul of SpeI
 +
273ul of 5M Tris
 +
 +
*The master mix for the (EcoRI and PstI) was;
 +
60ul of cut smart buffer
 +
5ul of XbaI
 +
5ul of SpeI
 +
278ul of 5M Tris
 +
 +
All 36 tubes were incubated overnight in 37 degrees room.
 +
 +
* Tomorrow the plasmids that were isolated from the miniprep would be run on a 1% agarose gel to distinguish between cells that have the pSB1C3/TOD genes from those that have the recircularised pSB1C3 vector.
 +
 +
* Meanwhile the plasmids from the miniprep were sent for sequencing this afternoon.
 +
 +
==Dissolution of polystyrene in Limonene==
 +
*This experiment was set up to decide how much limonene can economically dissolve polystyrene.
 +
 +
MATERIALS USED
 +
*20ml solution of 25% limonene.
 +
*8cm x 5cm x 2cm polystyrene block (thus volume of 80cm^3)
-
==Gel electrophoresis of isolated plasmids==
+
RESULT
-
* The plasmids that were isolated from the miniprep were run on a 1% agarose gel to confirm distinguish between cells that have the pSB1C3/TOD genes from those that have the recircularised pSB1C3 vector.
+
* It took ~33 minutes for the block to be completely dissolved

Latest revision as of 10:36, 10 September 2013

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Contents

Miniprep of overnight broth

  • Following on from 06/09/13 result,overnight broth was prepared yesterday from the single colonies that had grown on each of the 2 plates shown on 06/09/13.
  • The broth, which contains cells that have been transformed with pSB1C3/TOD genes, was minipreped using the Omega Bio-Tek kit.
  • This would enable us to confirm which cells have the recircularised plasmid and those with the ligated product.

Measurement of DNA concentration

Samples Volume (ul)Conc (ng/ul)280/260260/280
TOD X131.686.51.871.92
TOD X231.758.71.891.26
TOD X332.756.61.911.88
TOD X431.655.81.881.65
TOD X531.744.41.871.78
TOD X631.770.51.881.88
TOD F132.439.61.911.59
TOD F233.749.41.911.65
TOD F332.347.41.891.71
TOD F431.643.71.941.73
TOD F536.5571.891.50
TOD F63859.41.891.61
TOB G136.872.51.891.51
TOB G244.71.901.901.65
TOB G333.750.21.901.46
TOB G441.9551.931.35
TOB G534431.961.54
TOB G635481.891.72

DNA concentration was measured using Nanodrop.

Double Digestion of pSB1C3/TOD genes

  • The pSB1C3/TOD genes were double digested with (XbaI and SpeI) & (EcoRI and PstI)
  • The master mix for the (XbaI and SpeI) was;

60ul of cut smart buffer 5ul of XbaI 10ul of SpeI 273ul of 5M Tris

  • The master mix for the (EcoRI and PstI) was;

60ul of cut smart buffer 5ul of XbaI 5ul of SpeI 278ul of 5M Tris

All 36 tubes were incubated overnight in 37 degrees room.

  • Tomorrow the plasmids that were isolated from the miniprep would be run on a 1% agarose gel to distinguish between cells that have the pSB1C3/TOD genes from those that have the recircularised pSB1C3 vector.
  • Meanwhile the plasmids from the miniprep were sent for sequencing this afternoon.

Dissolution of polystyrene in Limonene

  • This experiment was set up to decide how much limonene can economically dissolve polystyrene. 
MATERIALS USED
  • 20ml solution of 25% limonene.
  • 8cm x 5cm x 2cm polystyrene block (thus volume of 80cm^3) 
RESULT
  • It took ~33 minutes for the block to be completely dissolved
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