Imperial College/4 September 2008

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===Cloning===
===Cloning===
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*Pfu PCR reactions for LacI gene, Aad9 gene and AmyE 3' Integration Sequence set up using the standard conditions and reagents [http://openwetware.org/wiki/IGEM:IMPERIAL/2008/Prototype/Wetlab/PCR]; LacI and AmyE 3' at 52°C for the first 10 cycles and 65°C for the last 20, Aad9 at 52°C then 60°C. 5μl of each PCR reaction run on a gel - results below
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*Pfu PCR reactions for LacI gene, Aad9 gene and AmyE 3' Integration Sequence were set up using the standard conditions and reagents [http://openwetware.org/wiki/IGEM:IMPERIAL/2008/Prototype/Wetlab/PCR]; LacI and AmyE 3' at 52°C for the first 10 cycles and 65°C for the last 20, Aad9 at 52°C then 60°C. 5μl of each PCR reaction run on a gel. Results are shown below.
*CAT biobrick digested with ''EcoRI'' and ''SpeI'' ready for assembly with the terminator
*CAT biobrick digested with ''EcoRI'' and ''SpeI'' ready for assembly with the terminator
*Termiantor biobrick cut with ''EcoRI'' and ''XbaI'' ready for assembly with CAT
*Termiantor biobrick cut with ''EcoRI'' and ''XbaI'' ready for assembly with CAT
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As can be seen, both the AmyE 3' integration sequence and the Aad9 (spectinomycin resistance) gene amplified properly. However, LacI did not and therefore, needs to be repeated.
As can be seen, both the AmyE 3' integration sequence and the Aad9 (spectinomycin resistance) gene amplified properly. However, LacI did not and therefore, needs to be repeated.
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===Preparation of Electrocompetent ''E. coli'' Cells===
===Preparation of Electrocompetent ''E. coli'' Cells===

Revision as of 23:09, 7 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]

4 September 2008

Wetlab

Cloning

  • Pfu PCR reactions for LacI gene, Aad9 gene and AmyE 3' Integration Sequence were set up using the standard conditions and reagents [http://openwetware.org/wiki/IGEM:IMPERIAL/2008/Prototype/Wetlab/PCR]; LacI and AmyE 3' at 52°C for the first 10 cycles and 65°C for the last 20, Aad9 at 52°C then 60°C. 5μl of each PCR reaction run on a gel. Results are shown below.
  • CAT biobrick digested with EcoRI and SpeI ready for assembly with the terminator
  • Termiantor biobrick cut with EcoRI and XbaI ready for assembly with CAT
  • GFP-Termiantor biobrick cut again with XbaI and SpeI to produce biobrick vectors for our PCR clones
  • GFP-Termiantor biobrick cut again with EcoRI and SpeI to produce biobrick vectors for our GeneArt produced clones

Results

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, lane 1 is the AmyE 3' Integration sequence, lane 2 the LacI and lane 3 the Aad9 PCR reaction

As can be seen, both the AmyE 3' integration sequence and the Aad9 (spectinomycin resistance) gene amplified properly. However, LacI did not and therefore, needs to be repeated.

Preparation of Electrocompetent E. coli Cells

We have prepared 42 aliquots of electrocompetent XL1 Blue E. coli cells using [http://openwetware.org/wiki/IGEM:IMPERIAL/2008/New/Protocols/XL1-Blue_preparation the relevant protocol] listed on our Protocols Page. The protocol was updated to include a higher innoculation value from the overnight culture.


Dry Lab

Motility

  • Tested Volocity software on 64-Bit analysis computer in SAF (FILM facility). Visually, volocity tracks cells very well, including synthetic data with a single motile ellipse without any change in shape. However, the software is unable to continuously track synthetic data of multiple ellipses with changing shape. It loses track of the cells after a few frames. When tested on real data, it tracks cells which run for fairly long, but it does not track tumbling cells. Most tracks do not last over 30 frames, which is considerable little for any form of good data to be extracted.
  • Hence we are still pursuing manual tracking, but if time permits, we will attempt to use other forms of filtering to achieve the best form of tracking for Volocity.
  • Error bars were plotted for Volocity software and uploaded onto the OWW wiki.
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