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| | #PCR with ''B.subtilis'' '''genomic''' DNA using AmyE Fw1 and R1 primers | | #PCR with ''B.subtilis'' '''genomic''' DNA using AmyE Fw1 and R1 primers |
| | #'''Single Colony PCR''' using AmyE Fw1 and R1 primers and AmyE Fw1 and R2 primers | | #'''Single Colony PCR''' using AmyE Fw1 and R1 primers and AmyE Fw1 and R2 primers |
| - | For each of these reactions a gradient of temperatures was used during the annealing stage of PCR, in summary the following reactions were carried out: | + | For each of these reactions a gradient of temperatures was used during the 10 cycles in the annealing stage of PCR and also the following 20 cycles, [[http://www.openwetware.org/index.php?title=IGEM:IMPERIAL/2008/Prototype/Wetlab/PCR| see our protocol for more information]] in summary the following reactions were carried out: |
| - | * 1. '''Vector''' - | + | * 1. '''Vector''' - 10 cycles at 57<sup>o</sup>C, 20 cycles at 64<sup>o</sup>C, Primers = Fw1/R1 |
| - | * 2. '''Vector''' - | + | * 2. '''Vector''' - 10 cycles at 55<sup>o</sup>C, 20 cycles at 62<sup>o</sup>C, Primers = Fw1/R1 |
| - | * 3. '''Vector''' - | + | * 3. '''Vector''' - 10 cycles at 53<sup>o</sup>C, 20 cycles at 60<sup>o</sup>C, Primers = Fw1/R1 |
| - | * 4. '''Genomic''' - | + | * 4. '''Genomic''' - 10 cycles at 57<sup>o</sup>C, 20 cycles at 64<sup>o</sup>C, Primers = Fw1/R1 |
| - | * 5. '''Genomic''' - | + | * 5. '''Genomic''' - 10 cycles at 55<sup>o</sup>C, 20 cycles at 62<sup>o</sup>C, Primers = Fw1/R1 |
| - | * 6. '''Genomic''' - | + | * 6. '''Genomic''' - 10 cycles at 53<sup>o</sup>C, 20 cycles at 60<sup>o</sup>C, Primers = Fw1/R1 |
| - | * 7. '''Single Colony PCR''' - | + | * 7. '''Single Colony PCR''' - 10 cycles at 52<sup>o</sup>C, 20 cycles at 64<sup>o</sup>C, Primers = Fw1/R1, Sample of colony 1ul |
| - | * 8. '''Single Colony PCR''' - | + | * 8. '''Single Colony PCR''' - 10 cycles at 50<sup>o</sup>C, 20 cycles at 64<sup>o</sup>C, Primers = Fw1/R1, Sample of colony 1ul |
| - | * 9. '''Single Colony PCR''' - | + | * 9. '''Single Colony PCR''' - 10 cycles at 48<sup>o</sup>C, 20 cycles at 64<sup>o</sup>C, Primers = Fw1/R1, Sample of colony 1ul |
| - | * 10. '''Single Colony PCR''' - | + | * 10. '''Single Colony PCR''' - 10 cycles at 52<sup>o</sup>C, 20 cycles at 64<sup>o</sup>C, Primers = Fw1/R1,Sample of colony 2ul |
| - | * 11. '''Single Colony PCR''' - | + | * 11. '''Single Colony PCR''' - 10 cycles at 50<sup>o</sup>C, 20 cycles at 64<sup>o</sup>C, Primers = Fw1/R1,Sample of colony 2ul |
| - | * 12. '''Single Colony PCR''' - | + | * 12. '''Single Colony PCR''' - 10 cycles at 48<sup>o</sup>C, 20 cycles at 64<sup>o</sup>C, Primers = Fw1/R1, Sample of colony 2ul |
|
| July
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| M | T | W | T | F | S | S
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| [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]
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| [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]
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| [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]
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| [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]
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| August
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| M | T | W | T | F | S | S
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| [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]
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| [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]
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| [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]
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| [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]
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| [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]
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| September
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| M | T | W | T | F | S | S
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| [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]
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| [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]
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| [http://2008.igem.org/Imperial_College/29_September_2008 29]
| [http://2008.igem.org/Imperial_College/30_September_2008 30]
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29 August 2008
WetLab
Midipreps
- Digested Midipreps of Biobricks - The midipreps of the following constructs were digested with EcoRI and SpeI in order to confirm the expected length of the biobrick. The following biobricks were midi preped, digested and analysed on agarose gel electrophoresis:
- Terminator, size of biobrick =
- AK3, size of biobrick =
- The gel below shows the results of this analysis,
 Lanes : M = Marker, 1 = Terminator Midi 1 Undigested, 2 = Terminator Midi 1 Digested, 3 = Terminator Midi 2 Undigested, 4 = Terminator Midi 2 Digested, 5 = AK3 Midi 1 Undigested, 6 = AK3 Midi 1 Digested, 7 = AK3 Midi 2 Undigested, 8 = AK3 Midi 2 Digested, PCR
In a pursuit to verify the various PCR protocols and PCR primers we carried out the following reactions:
- PCR with the pDR110 Vector using AmyE Fw1 and R1 primers
- PCR with B.subtilis genomic DNA using AmyE Fw1 and R1 primers
- Single Colony PCR using AmyE Fw1 and R1 primers and AmyE Fw1 and R2 primers
For each of these reactions a gradient of temperatures was used during the 10 cycles in the annealing stage of PCR and also the following 20 cycles, see our protocol for more information in summary the following reactions were carried out:
- 1. Vector - 10 cycles at 57oC, 20 cycles at 64oC, Primers = Fw1/R1
- 2. Vector - 10 cycles at 55oC, 20 cycles at 62oC, Primers = Fw1/R1
- 3. Vector - 10 cycles at 53oC, 20 cycles at 60oC, Primers = Fw1/R1
- 4. Genomic - 10 cycles at 57oC, 20 cycles at 64oC, Primers = Fw1/R1
- 5. Genomic - 10 cycles at 55oC, 20 cycles at 62oC, Primers = Fw1/R1
- 6. Genomic - 10 cycles at 53oC, 20 cycles at 60oC, Primers = Fw1/R1
- 7. Single Colony PCR - 10 cycles at 52oC, 20 cycles at 64oC, Primers = Fw1/R1, Sample of colony 1ul
- 8. Single Colony PCR - 10 cycles at 50oC, 20 cycles at 64oC, Primers = Fw1/R1, Sample of colony 1ul
- 9. Single Colony PCR - 10 cycles at 48oC, 20 cycles at 64oC, Primers = Fw1/R1, Sample of colony 1ul
- 10. Single Colony PCR - 10 cycles at 52oC, 20 cycles at 64oC, Primers = Fw1/R1,Sample of colony 2ul
- 11. Single Colony PCR - 10 cycles at 50oC, 20 cycles at 64oC, Primers = Fw1/R1,Sample of colony 2ul
- 12. Single Colony PCR - 10 cycles at 48oC, 20 cycles at 64oC, Primers = Fw1/R1, Sample of colony 2ul
|
"
