Imperial College/8 September 2008

From 2008.igem.org

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**1 cycle 95<sup>o</sup>C 30 seconds
**1 cycle 95<sup>o</sup>C 30 seconds
**30 cycles, 95<sup>o</sup>C 30 seconds, 58/56<sup>o</sup>C 1 minute, 72<sup>o</sup>C for 2 minutes and 30 seconds.
**30 cycles, 95<sup>o</sup>C 30 seconds, 58/56<sup>o</sup>C 1 minute, 72<sup>o</sup>C for 2 minutes and 30 seconds.
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[[Image:8-08.PNG|thumb|400px|center|A 1% Agarose gel showing the results of various PCR reactions. Each lane is loaded with 5ul of PCR reaction and 1ul of 6x sample buffer. M = Marker]]
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Upper Gel
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Lane 1 -
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Lane 2 -
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Lane 3 -
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Lane 4 -
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Lane 5 -
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Lane 6 -
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Lane 7 - pSB primers, biobrick template, 60<sup>o</sup>C
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Lane 8 - pSB primers, biobrick template, 58<sup>o</sup>C
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Lane 9 - pSB primers, biobrick template, 56<sup>o</sup>C
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Lane 10 - LacI primers, Pfu polymerase and pDR11 template. 56<sup>o</sup>C for 10 cycles then 64<sup>o</sup>C for 20 cycles.
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Lower Gel
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Lane 1 - pSB1A2 cut with ''Eco''RI and ''Spe''I
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Lane 2 - construct 7 cut with ''Eco''RI and ''Spe''I
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Lane 3 - construct 8 cut with ''Eco''RI and ''Spe''I
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Lane 4 - construct 9 cut with ''Eco''RI and ''Spe''I
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Lane 5 - construct 11 cut with ''Eco''RI and ''Spe''I
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Lane 6 - pSB1AK3 cut with ''Eco''RI and ''Spe''I
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Lane 7 - CAT insert (cut ''Eco''RI - ''Spe''I)
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Lane 8 - Terminator vector (cut ''Eco''RI - ''Xba''I)
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Lane 9 - pSB1A2 cut with ''Xba''I and ''Spe''I
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Lane 10 - AmyE 5' IS clone (cut ''Xba''I - ''Spe''I)
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Lane 11 - Aad9 clone (cut ''Xba''I - ''Spe''I)
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Lane 12 - AmyE 3' IS clone (cut ''Xba''I - ''Spe''I)
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Lane 13 - pSB1AK3 cut with ''Xba''I and ''Spe''I
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The results of this gel were used to calculate the amount of reagents were required for our ligations
{{Imperial/EndPage}}
{{Imperial/EndPage}}

Revision as of 00:00, 12 September 2008

August
MTWTFSS
        [http://2008.igem.org/Imperial_College/1_August_2008 1] [http://2008.igem.org/Imperial_College/2_August_2008 2] [http://2008.igem.org/wiki/index.php?title=Imperial_College/3_August_2008&action=edit 3]
[http://2008.igem.org/Imperial_College/4_August_2008 4] [http://2008.igem.org/Imperial_College/5_August_2008 5] [http://2008.igem.org/wiki/index.php?title=Imperial_College/6_August_2008&action=edit 6] [http://2008.igem.org/Imperial_College/7_August_2008 7] [http://2008.igem.org/Imperial_College/8_August_2008 8] [http://2008.igem.org/wiki/index.php?title=Imperial_College/9_August_2008&action=edit 9] [http://2008.igem.org/Imperial_College/10_August_2008 10]
[http://2008.igem.org/Imperial_College/11_August_2008 11] [http://2008.igem.org/Imperial_College/12_August_2008 12] [http://2008.igem.org/Imperial_College/13_August_2008 13] [http://2008.igem.org/Imperial_College/14_August_2008 14] [http://2008.igem.org/Imperial_College/15_August_2008 15] [http://2008.igem.org/Imperial_College/16_August_2008 16] [http://2008.igem.org/Imperial_College/17_August_2008 17]
[http://2008.igem.org/Imperial_College/18_August_2008 18] [http://2008.igem.org/Imperial_College/19_August_2008 19] [http://2008.igem.org/Imperial_College/20_August_2008 20] [http://2008.igem.org/Imperial_College/21_August_2008 21] [http://2008.igem.org/Imperial_College/22_August_2008 22] [http://2008.igem.org/wiki/index.php?title=Imperial_College/23_August_2008&action=edit 23] [http://2008.igem.org/Imperial_College/24_August_2008 24]
[http://2008.igem.org/Imperial_College/25_August_2008 25] [http://2008.igem.org/Imperial_College/26_August_2008 26] [http://2008.igem.org/Imperial_College/27_August_2008 27] [http://2008.igem.org/Imperial_College/28_August_2008 28] [http://2008.igem.org/Imperial_College/29_August_2008 29] [http://2008.igem.org/wiki/index.php?title=Imperial_College/30_August_2008&action=edit 30] [http://2008.igem.org/Imperial_College/31_August_2008 31]
September
MTWTFSS
[http://2008.igem.org/Imperial_College/1_September_2008 1] [http://2008.igem.org/Imperial_College/2_September_2008 2] [http://2008.igem.org/Imperial_College/3_September_2008 3] [http://2008.igem.org/Imperial_College/4_September_2008 4] [http://2008.igem.org/Imperial_College/5_September_2008 5] [http://2008.igem.org/wiki/index.php?title=Imperial_College/6_September_2008&action=edit 6] [http://2008.igem.org/Imperial_College/7_September_2008 7]
[http://2008.igem.org/Imperial_College/8_September_2008 8] [http://2008.igem.org/Imperial_College/9_September_2008 9] [http://2008.igem.org/Imperial_College/10_September_2008 10] [http://2008.igem.org/Imperial_College/11_September_2008 11] [http://2008.igem.org/Imperial_College/12_September_2008 12] [http://2008.igem.org/wiki/index.php?title=Imperial_College/13_September_2008&action=edit 13] [http://2008.igem.org/wiki/index.php?title=Imperial_College/14_September_2008&action=edit 14]
[http://2008.igem.org/Imperial_College/15_September_2008 15] [http://2008.igem.org/Imperial_College/16_September_2008 16] [http://2008.igem.org/Imperial_College/17_September_2008 17] [http://2008.igem.org/Imperial_College/18_September_2008 18] [http://2008.igem.org/Imperial_College/19_September_2008 19] [http://2008.igem.org/wiki/index.php?title=Imperial_College/20_September_2008&action=edit 20] [http://2008.igem.org/wiki/index.php?title=Imperial_College/21_September_2008&action=edit 21]
[http://2008.igem.org/Imperial_College/22_September_2008 22] [http://2008.igem.org/Imperial_College/23_September_2008 23] [http://2008.igem.org/Imperial_College/24_September_2008 24] [http://2008.igem.org/Imperial_College/25_September_2008 25] [http://2008.igem.org/Imperial_College/26_September_2008 26] [http://2008.igem.org/wiki/index.php?title=Imperial_College/27_September_2008&action=edit 27] [http://2008.igem.org/wiki/index.php?title=Imperial_College/28_September_2008&action=edit 28]
[http://2008.igem.org/Imperial_College/29_September_2008 29] [http://2008.igem.org/Imperial_College/30_September_2008 30]
October
MTWTFSS
    [http://2008.igem.org/Imperial_College/1_October_2008 1] [http://2008.igem.org/Imperial_College/2_October_2008 2] [http://2008.igem.org/wiki/index.php?title=Imperial_College/3_October_2008&action=edit 3] [http://2008.igem.org/wiki/index.php?title=Imperial_College/4_October_2008&action=edit 4] [http://2008.igem.org/wiki/index.php?title=Imperial_College/5_October_2008&action=edit 5]
[http://2008.igem.org/wiki/index.php?title=Imperial_College/6_October_2008&action=edit 6] [http://2008.igem.org/Imperial_College/7_October_2008 7] [http://2008.igem.org/wiki/index.php?title=Imperial_College/8_October_2008&action=edit 8] [http://2008.igem.org/Imperial_College/9_October_2008 9] [http://2008.igem.org/wiki/index.php?title=Imperial_College/10_October_2008&action=edit 10] [http://2008.igem.org/wiki/index.php?title=Imperial_College/11_October_2008&action=edit 11] [http://2008.igem.org/wiki/index.php?title=Imperial_College/12_October_2008&action=edit 12]
[http://2008.igem.org/Imperial_College/13_October_2008 13] [http://2008.igem.org/wiki/index.php?title=Imperial_College/14_October_2008&action=edit 14] [http://2008.igem.org/Imperial_College/15_October_2008 15] [http://2008.igem.org/Imperial_College/16_October_2008 16] [http://2008.igem.org/Imperial_College/17_October_2008 17] [http://2008.igem.org/wiki/index.php?title=Imperial_College/18_October_2008&action=edit 18] [http://2008.igem.org/wiki/index.php?title=Imperial_College/19_October_2008&action=edit 19]
[http://2008.igem.org/wiki/index.php?title=Imperial_College/20_October_2008&action=edit 20] [http://2008.igem.org/wiki/index.php?title=Imperial_College/21_October_2008&action=edit 21] [http://2008.igem.org/wiki/index.php?title=Imperial_College/22_October_2008&action=edit 22] [http://2008.igem.org/wiki/index.php?title=Imperial_College/23_October_2008&action=edit 23] [http://2008.igem.org/wiki/index.php?title=Imperial_College/24_October_2008&action=edit 24] [http://2008.igem.org/wiki/index.php?title=Imperial_College/25_October_2008&action=edit 25] [http://2008.igem.org/wiki/index.php?title=Imperial_College/26_October_2008&action=edit 26]
[http://2008.igem.org/wiki/index.php?title=Imperial_College/27_October_2008&action=edit 27] [http://2008.igem.org/wiki/index.php?title=Imperial_College/28_October_2008&action=edit 28] [http://2008.igem.org/wiki/index.php?title=Imperial_College/29_October_2008&action=edit 29] [http://2008.igem.org/wiki/index.php?title=Imperial_College/30_October_2008&action=edit 30] [http://2008.igem.org/wiki/index.php?title=Imperial_College/31_October_2008&action=edit 31]

8 September 2008

Dry Lab

Motility

  • Assessed error with data extraction algorithm which extracts run velocity, run duration, tumbling angle and tumbling duration. Following steps were taken (and documented on OWW Wiki):
    1. Used known synthetic trajectory as an input for data extraction algorithm
    2. After obtaining motility data on the above 4 characteristics, posteriors were plotted. mu and sigma for run velocity, lambda for run and tumbling duration.
    3. Error assessed by finding % error between parameter value for which posterior is maximised and the actual value of the parameter used to generate synthetic trajectory.
  • It was found that the errors are quite large. This is due to the low frame rate such that tumbles which are less than 1/(frame rate) do not cause the cell to be stationary. Only way to get around this problem with real data is to increase the frame rate at which our videos are captured.
  • We also tracked more cells today, tip and centre. Total number of cells tracked so far is 20, with over 5000 data points (coordinates).
  • Segmentation algorithm still does not provide us with satisfactory visual segmentation. Clustering software however, can find 2 centres, but we are not convinced whether this is of any significance.
  • This is an important issue to be resolved before we can even move onto modelling motility of bacteria.

Wet Lab

Transformation

  • Today we confirmed that the transformation has worked! A single colony PCR confirmed that the integration of the vector pDR110 was successful. The conditions set up were as follows:
    • Single colony PCR using genomic (control) B.subtilis
    • Single colony PCR using transformed B.subtilis.
  • The following settings were used:
    • 1 cycle 95oC 30 seconds
    • 30 cycles, 95oC 30 seconds, 58/56oC 1 minute, 72oC for 2 minutes and 30 seconds.
A 1% Agarose gel showing the results of various PCR reactions. Each lane is loaded with 5ul of PCR reaction and 1ul of 6x sample buffer. M = Marker

Upper Gel

Lane 1 -

Lane 2 -

Lane 3 -

Lane 4 -

Lane 5 -

Lane 6 -

Lane 7 - pSB primers, biobrick template, 60oC

Lane 8 - pSB primers, biobrick template, 58oC

Lane 9 - pSB primers, biobrick template, 56oC

Lane 10 - LacI primers, Pfu polymerase and pDR11 template. 56oC for 10 cycles then 64oC for 20 cycles.

Lower Gel


Lane 1 - pSB1A2 cut with EcoRI and SpeI

Lane 2 - construct 7 cut with EcoRI and SpeI

Lane 3 - construct 8 cut with EcoRI and SpeI

Lane 4 - construct 9 cut with EcoRI and SpeI

Lane 5 - construct 11 cut with EcoRI and SpeI

Lane 6 - pSB1AK3 cut with EcoRI and SpeI

Lane 7 - CAT insert (cut EcoRI - SpeI)

Lane 8 - Terminator vector (cut EcoRI - XbaI)

Lane 9 - pSB1A2 cut with XbaI and SpeI

Lane 10 - AmyE 5' IS clone (cut XbaI - SpeI)

Lane 11 - Aad9 clone (cut XbaI - SpeI)

Lane 12 - AmyE 3' IS clone (cut XbaI - SpeI)

Lane 13 - pSB1AK3 cut with XbaI and SpeI

The results of this gel were used to calculate the amount of reagents were required for our ligations


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