Team:Newcastle/6 September 2010

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====PHOTOS====
====PHOTOS====
 +
 +
=Hyperspankoid characterisation=
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 +
==Aims==
 +
The aim of these experiments is to ligate the promoters hyperspankoid, pspacoid and hyperspank in front of RFP of the pSB1C3 plasmid. In order to do this, the first step is to amplify the three promoter sequences and the pSB1C3 plasmid using Phusion PCR and the different primers and template DNA.
 +
 +
==Materials and Protocol==
 +
Please refer to [[Team:Newcastle/PCR#Phusion_PCR| Phusion PCR]] for Phusion PCR protocol.
 +
 +
The details for the four PCR reactions are included in the table below:
 +
 +
{|border=1
 +
|-
 +
!'''Tube'''
 +
!'''Part to be amplified'''
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!'''DNA fragment consisting the part'''
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!'''Forward primer'''
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!'''Reverse Primer'''
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!'''Melting Temperature (Tm in °C) '''
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!'''Size of the fragment (in bp)'''
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!'''Extension time* (in seconds)'''
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|-
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|1
 +
|Hyperspankoid 
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|
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|Prom_for
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|HpSpanPspacoid_rev
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|53.3 (53)
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|2046 +
 +
|70
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|-
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|2
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|Pspacoid
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|
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|Prom_for
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|HpSpanPspacoid_rev
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|51.7 (51)
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|139 +
 +
|15
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|-
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|3
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|Hyperspank
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|
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|Prom_for
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|Pspac-hy rev
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|53.0 (53)
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|2753 +
 +
|110
 +
|-
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|4
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|Plasmid
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|vector pSB1C3/RFP
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|Vector/RFP_for
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|P2-V1
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|50.9 (51)
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|116 +
 +
|15
 +
|}
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 +
'''Table 2''': This table shows the four different Phusion PCR reactions that were carried out today. If this is successful, the four parts will be ligated together for the construction of subtilin immunity BioBrick with the help of Gibson Cloning method.
 +
* The extension rate of the Phusion polymerase is 1 Kb/ 30 seconds. Thus the extension time of each and every PCR reaction is slightly different.
 +
PCR reactions - Hyperspankoid - Prom_For and HpSpanPspacoid rev - using yneA as template - Tm 65 - Elongation 15 secs
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 +
- Pspacoid - Prom_For and HpSpanPspacoid rev - using kinA as tempate - Tm 65 - Elongation 15 secs
 +
 +
- Hyperspank - Prom_for and Pspac-hy rev - using K143055 from Bacillus plate - BS022, I18 - Tm 62 - Elongation 15 secs
 +
 +
- Plasmid - Vector/RFP for and P2-V1 - using pSB1C3 - Tm 64 - Elongation 60 secs
{{Team:Newcastle/footer}}
{{Team:Newcastle/footer}}

Revision as of 09:13, 8 September 2010

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Contents

rocF

Aims

rocF miniprep: we aim to purify rocF in p.....

Materials and protocol

See Mini prep protocol

Results

Nanodrop

rocF

Concentration of DNA in ng/µl

  1. 93.1
  2. 102.0
  3. 99.4
  4. 101.0
  5. 98.8
  6. 104.7
  7. 93.1
  8. 57.9
  9. 81.9


Subtilin Immunity BioBrick

Aims

Overnight cultures of all the colonies from the 'SI 1/9/10' plates was carried out today.

Methods

The Growing Overnight Cultures protocol was followed. The colonies were grown on LB with chloramphenicol. Altogether 16 colonies were produced on the plates and were left overnight at 37°C.

Sucrose/Levan's glue

The sucrose cultures and controls were compared. The cultures grown on sucrose should be considerably thicker. The overnight cultures of the different strains were also grown on solid agar plates with and without sucrose.

PHOTOS

Hyperspankoid characterisation

Aims

The aim of these experiments is to ligate the promoters hyperspankoid, pspacoid and hyperspank in front of RFP of the pSB1C3 plasmid. In order to do this, the first step is to amplify the three promoter sequences and the pSB1C3 plasmid using Phusion PCR and the different primers and template DNA.

Materials and Protocol

Please refer to Phusion PCR for Phusion PCR protocol.

The details for the four PCR reactions are included in the table below:

Tube Part to be amplified DNA fragment consisting the part Forward primer Reverse Primer Melting Temperature (Tm in °C) Size of the fragment (in bp) Extension time* (in seconds)
1 Hyperspankoid Prom_for HpSpanPspacoid_rev 53.3 (53) 2046 + 70
2 Pspacoid Prom_for HpSpanPspacoid_rev 51.7 (51) 139 + 15
3 Hyperspank Prom_for Pspac-hy rev 53.0 (53) 2753 + 110
4 Plasmid vector pSB1C3/RFP Vector/RFP_for P2-V1 50.9 (51) 116 + 15

Table 2: This table shows the four different Phusion PCR reactions that were carried out today. If this is successful, the four parts will be ligated together for the construction of subtilin immunity BioBrick with the help of Gibson Cloning method.

  • The extension rate of the Phusion polymerase is 1 Kb/ 30 seconds. Thus the extension time of each and every PCR reaction is slightly different.

PCR reactions - Hyperspankoid - Prom_For and HpSpanPspacoid rev - using yneA as template - Tm 65 - Elongation 15 secs

- Pspacoid - Prom_For and HpSpanPspacoid rev - using kinA as tempate - Tm 65 - Elongation 15 secs

- Hyperspank - Prom_for and Pspac-hy rev - using K143055 from Bacillus plate - BS022, I18 - Tm 62 - Elongation 15 secs

- Plasmid - Vector/RFP for and P2-V1 - using pSB1C3 - Tm 64 - Elongation 60 secs

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