Imperial College/3 September 2008

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*A negative control was used at 56<sup>o</sup>C where no genomic DNA was added.The results are shown in figure 1.1 below.
*A negative control was used at 56<sup>o</sup>C where no genomic DNA was added.The results are shown in figure 1.1 below.
<br>
<br>
-
*To confirm if the single colony PCR protocol is working we used this protocol with a new set of primers and a set that has been primers shown to work with purified genomic DNA. AmyE fw1 and rv1 (shown to work) are primers used to purify the 5' integration site from ''B.subtilis'' and AmyE fw2 and rv2 (not tested yet) are primers used to purify the 3'integration sites from ''B.subtilis''. We performed the following reactions,
+
*To confirm if the single colony PCR protocol is working we used this protocol with a new set of primers and a set that has been primers shown to work with purified genomic DNA. AmyE fw1 and rv1 (shown to work) are primers used to purify the 5' integration site from ''B.subtilis'' and AmyE fw2 and rv2 (not tested yet) are primers used to purify the 3'integration sites from ''B.subtilis''. We performed the single colony PCR with Fw1 and Rv2 (not tested) and Fw1 and Rv1 (tested) under the following conditions:
-
**
+
*Using the primer, Fw1 and Rv2 -
-
 
+
**1 cycle 95<sup>o</sup>C for 30 seconds,
-
 
+
**10 cycles, 30 seconds 95<sup>o</sup>C, 1 minute at 52/50/48<sup>o</sup>C, 5 minutes at 72<sup>o</sup>C
-
 
+
**20 cycles, 30 seconds 95<sup>o</sup>C, 1 minute at 61<sup>o</sup>C, 5 minutes at 72<sup>o</sup>C
 +
**1 cycle, 1 minute at 72<sup>o</sup>C.
 +
*Using the primers, Fw1 and Rv1 -
 +
**1 cycle 95<sup>o</sup>C for 30 seconds,
 +
**10 cycles, 30 seconds 95<sup>o</sup>C, 1 minute at 48<sup>o</sup>C, 5 minutes at 72<sup>o</sup>C
 +
**20 cycles, 30 seconds 95<sup>o</sup>C, 1 minute at 61<sup>o</sup>C, 5 minutes at 72<sup>o</sup>C
 +
**1 cycle, 1 minute at 72<sup>o</sup>C.
 +
*A negative control was also used where no primers were added.
===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.]]

Revision as of 08:29, 5 September 2008

July
MTWTFSS
  [http://2008.igem.org/wiki/index.php?title=Imperial_College/1_July_2008&action=edit 1] [http://2008.igem.org/wiki/index.php?title=Imperial_College/2_July_2008&action=edit 2] [http://2008.igem.org/wiki/index.php?title=Imperial_College/3_July_2008&action=edit 3] [http://2008.igem.org/wiki/index.php?title=Imperial_College/4_July_2008&action=edit 4] [http://2008.igem.org/wiki/index.php?title=Imperial_College/5_July_2008&action=edit 5] [http://2008.igem.org/wiki/index.php?title=Imperial_College/6_July_2008&action=edit 6]
[http://2008.igem.org/Imperial_College/7_July_2008 7] [http://2008.igem.org/Imperial_College/8_July_2008 8] [http://2008.igem.org/Imperial_College/9_July_2008 9] [http://2008.igem.org/wiki/index.php?title=Imperial_College/10_July_2008&action=edit 10] [http://2008.igem.org/Imperial_College/11_July_2008 11] [http://2008.igem.org/wiki/index.php?title=Imperial_College/12_July_2008&action=edit 12] [http://2008.igem.org/wiki/index.php?title=Imperial_College/13_July_2008&action=edit 13]
[http://2008.igem.org/wiki/index.php?title=Imperial_College/14_July_2008&action=edit 14] [http://2008.igem.org/Imperial_College/15_July_2008 15] [http://2008.igem.org/Imperial_College/16_July_2008 16] [http://2008.igem.org/wiki/index.php?title=Imperial_College/17_July_2008&action=edit 17] [http://2008.igem.org/Imperial_College/18_July_2008 18] [http://2008.igem.org/wiki/index.php?title=Imperial_College/19_July_2008&action=edit 19] [http://2008.igem.org/wiki/index.php?title=Imperial_College/20_July_2008&action=edit 20]
[http://2008.igem.org/Imperial_College/21_July_2008 21] [http://2008.igem.org/Imperial_College/22_July_2008 22] [http://2008.igem.org/Imperial_College/23_July_2008 23] [http://2008.igem.org/Imperial_College/24_July_2008 24] [http://2008.igem.org/Imperial_College/25_July_2008 25] [http://2008.igem.org/wiki/index.php?title=Imperial_College/26_July_2008&action=edit 26] [http://2008.igem.org/wiki/index.php?title=Imperial_College/27_July_2008&action=edit 27]
[http://2008.igem.org/Imperial_College/28_July_2008 28] [http://2008.igem.org/Imperial_College/29_July_2008 29] [http://2008.igem.org/Imperial_College/30_July_2008 30] [http://2008.igem.org/wiki/index.php?title=Imperial_College/31_July_2008&action=edit 31]
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]

Wetlab

Transformation of B.subtilis

  • Previously we have tried to test efficient integration into the B.subtilis genome. However, the primers we were using have so far failed to yield either a positive or negative result when a signle colony PCR was carried out on transformed B.subtilis. In order to check firstly if the primers are working and secondly if the single colony PCR protocol is working we carried out a series of testing. First to test the verification primers we are going to carry out a PCR on purified genome, that we have previously used successfully in a PCR reaction. We set up the following conditions:
    • 1 cycle 95oC for 30 seconds
    • 30 cycles, 30 seconds 95oC, 1 minute at 60/58/56oC, 5 minutes at 72oC
  • A negative control was used at 56oC where no genomic DNA was added.The results are shown in figure 1.1 below.


  • To confirm if the single colony PCR protocol is working we used this protocol with a new set of primers and a set that has been primers shown to work with purified genomic DNA. AmyE fw1 and rv1 (shown to work) are primers used to purify the 5' integration site from B.subtilis and AmyE fw2 and rv2 (not tested yet) are primers used to purify the 3'integration sites from B.subtilis. We performed the single colony PCR with Fw1 and Rv2 (not tested) and Fw1 and Rv1 (tested) under the following conditions:
  • Using the primer, Fw1 and Rv2 -
    • 1 cycle 95oC for 30 seconds,
    • 10 cycles, 30 seconds 95oC, 1 minute at 52/50/48oC, 5 minutes at 72oC
    • 20 cycles, 30 seconds 95oC, 1 minute at 61oC, 5 minutes at 72oC
    • 1 cycle, 1 minute at 72oC.
  • Using the primers, Fw1 and Rv1 -
    • 1 cycle 95oC for 30 seconds,
    • 10 cycles, 30 seconds 95oC, 1 minute at 48oC, 5 minutes at 72oC
    • 20 cycles, 30 seconds 95oC, 1 minute at 61oC, 5 minutes at 72oC
    • 1 cycle, 1 minute at 72oC.
  • A negative control was also used where no primers were added.

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.
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