Team:Groningen/Notebook/14 July 2009

From 2009.igem.org

(Difference between revisions)
(GVP Cluster)
(GVP Cluster)
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===GVP Cluster===
===GVP Cluster===
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[[Image:Plasmid Miniprep Kit Flow Protocol.gif|thumb|200px| www.sigmaaldrich.com]]
+
[[Image:Plasmid Miniprep Kit Flow Protocol.gif|thumb|150px| www.sigmaaldrich.com]]
'''Plasmid Purification'''
'''Plasmid Purification'''
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* To the mixture 200μL Lysis Solution was added, mixed by inverting the cup, and stored at room temperature for 5 minutes.
* To the mixture 200μL Lysis Solution was added, mixed by inverting the cup, and stored at room temperature for 5 minutes.
* 350μL of Neutralisation Solution was added and the tubes inverted.
* 350μL of Neutralisation Solution was added and the tubes inverted.
-
* Cell debri was pelleted by centrifugation at full speed for 1 min.
+
* Cell debri was pelleted by centrifugation at full speed for 10 min.
-
*  
+
* Column was prepared by adding 500μL Column Preparation Solution and centrifuging for 1 min. full speed
 +
* The lysate was transfered to the column and centrifuged for 1 min. full speed
 +
* Column was washed with both washing solutions
 +
* Plasmids were eluted with 30μL MQ and stored in the fridge
'''Concentration of Plasmids'''
'''Concentration of Plasmids'''

Revision as of 09:12, 14 July 2009

Igemhomelogo.png

Wet

GVP Cluster

www.sigmaaldrich.com

Plasmid Purification

Plasmid isolation was performed on the cultures of GVP and Terminator containing cells with the "Sygma-Aldrich™ GenElute™ Plasmid Miniprep Kit".

  • From each tube 5mL of culture was collected in a 2mL cup, and the cells were pelleted by centrifugation for 1 min. at max. speed and the supernatant discarded.
  • Cells were resuspended in 200μL Resuspension Solution by up and down pipetting.
  • To the mixture 200μL Lysis Solution was added, mixed by inverting the cup, and stored at room temperature for 5 minutes.
  • 350μL of Neutralisation Solution was added and the tubes inverted.
  • Cell debri was pelleted by centrifugation at full speed for 10 min.
  • Column was prepared by adding 500μL Column Preparation Solution and centrifuging for 1 min. full speed
  • The lysate was transfered to the column and centrifuged for 1 min. full speed
  • Column was washed with both washing solutions
  • Plasmids were eluted with 30μL MQ and stored in the fridge

Concentration of Plasmids

The concentration of isolated plasmid was determined with the use of a nano-drop.

GVP eluted in MQ

  • ng/μL
  • (260/280)
  • (260/230)

Transporters

Dry

April
MTWTFSS
    1 2 3 4 5
6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30
May
MTWTFSS
        1 2 3
4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30 31
June
MTWTFSS
1 2 3 4 5 6 7
8 9 10 11 12 13 14
15 16 17 18 19 20 21
22 23 24 25 26 27 28
29 30
July
MTWTFSS
    1 2 3 4 5
6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30 31
August
MTWTFSS
          1 2
3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30
31
September
MTWTFSS
  1 2 3 4 5 6
7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30
October
MTWTFSS
      1 2 3 4
5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30 31
November
MTWTFSS
            1
2 3 4 5 6 7 8
9 10 11 12 13 14 15
16 17 18 19 20 21 22
23 24 25 26 27 28 29
30