Imperial College/2 September 2008

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=2 September 2008=
==Wet Lab==
==Wet Lab==
 +
===Cloning===
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*Pfu PCR reactions for both AmyE integration sequences and the Laci gene were set up according to the standard [http://openwetware.org/index.php?title=IGEM:IMPERIAL/2008/Prototype/Wetlab/PCR protocol], using the optimal conditions determined yesterday and results run on a gel along with Aad9 Taq polymerase condiitons test (54<sup>o</sup>C, 52<sup>o</sup>C or 50<sup>o</sup>C for the first 10 cycles then 60<sup>o</sup>C for the last 20 cycles). Apropraite negativbe controls were used. For results, see tomorrow's entry.
 +
*XL1-Blue cells containing the constructs in plasmids from GeneArt were grown up and incoulated into an overnight culture for midiprep tomorrow
 +
===Transformation of ''B.subtilis''===
===Transformation of ''B.subtilis''===
*In order to confirm the transformations of ''B.subtilis'' that were performed in the previous weeks, we have designed validation primers. These validation primers are complementary to regions flanking the amyE locus and as a results the PCR product size will reflect any inserted DNA within this region. Using these primers a single colony PCR was performed, (link to prorotcol for single colony PCR for verficication.
*In order to confirm the transformations of ''B.subtilis'' that were performed in the previous weeks, we have designed validation primers. These validation primers are complementary to regions flanking the amyE locus and as a results the PCR product size will reflect any inserted DNA within this region. Using these primers a single colony PCR was performed, (link to prorotcol for single colony PCR for verficication.
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===Results===
===Results===
[[Image:RESULTS2.JPG|thumb|600px|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.]]
[[Image:RESULTS2.JPG|thumb|600px|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.]]
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{{Imperial/EndPage|Notebook|Notebook}}

Latest revision as of 20:43, 28 October 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]

2 September 2008

Wet Lab

Cloning

  • Pfu PCR reactions for both AmyE integration sequences and the Laci gene were set up according to the standard [http://openwetware.org/index.php?title=IGEM:IMPERIAL/2008/Prototype/Wetlab/PCR protocol], using the optimal conditions determined yesterday and results run on a gel along with Aad9 Taq polymerase condiitons test (54oC, 52oC or 50oC for the first 10 cycles then 60oC for the last 20 cycles). Apropraite negativbe controls were used. For results, see tomorrow's entry.
  • XL1-Blue cells containing the constructs in plasmids from GeneArt were grown up and incoulated into an overnight culture for midiprep tomorrow

Transformation of B.subtilis

  • In order to confirm the transformations of B.subtilis that were performed in the previous weeks, we have designed validation primers. These validation primers are complementary to regions flanking the amyE locus and as a results the PCR product size will reflect any inserted DNA within this region. Using these primers a single colony PCR was performed, (link to prorotcol for single colony PCR for verficication.
  • The results of this protocol are shown on figure 1 a 1% agarose gel. The lanes concerned are 1-4 and represent different annealing temperatures used, which were as follows:
    • 1-62 oC
    • 2-60 oC
    • 3-58 oC
    • 4-58 oC - Negative control - no DNA!!!
  • If integration of the pDR110 plasmid was efficient we should see a band at 5.5kb, and if it has failed we should see a band at 2.5kb. We do not see these bands. The reason for this is unknown and so we will carry out further experiments, firstly to validate the verification primers and secondly to validate the signle colony PCR technique. To do this we have used primers previuously shown to work in a single colony PCR protocol and used the verfification primers with purified genomic DNA which has also previously been shown to work as a template. The method and results for this will be posted tomorrow.

Testing of Primers

  • The following primers were tested today to confirm that they are working and if so the optimum annealing temperatures. The primers tested were the XylR and the EpsE 1 primers which were performed on purified genomic DNA. The protocol for this PCR reaction was following the standard protocol for PCR, click here
  • The results of this protocol are shown on figure 1 a 1% agarose gel. The lanes concerned are 1-4 and represent different annealing temperatures used, which were as follows:
    • 5-51 oC - XylR
    • 6-49 oC - XylR
    • 7-47 oC - XylR
    • 8-46 oC - XylR
    • 9-46 oC - Negative control - no DNA!!!
    • 10-50 oC - AmyE 1 positive control as previously shown to work.
    • 11-53 oC - EspE1
    • 12-51 oC - EspE1
    • 13-49 oC - EspE1
    • 14-47 oC - EspE1
    • 15-47 oC - Negative control - no DNA!!!
    • 16-51 oC - AmyE 1 positive control as previously shown to work.
  • Results - From the gel below it can be seen that the EpsE PCR reactions have worked, however with slight levels of contamination when a high annealing temperature is used. The XylR PCR reaction has not work efficiently, clearly visible from the faint bands. This means that the temperatures for annealing need to be optimised further.

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.