Team:Newcastle/10 August 2010

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=Gel extraction of ''rocF'' BioBrick components=
 
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==Gel Electrophoresis for Amplified Pspac_oid promoter, Double terminator and Plasmid Vector pSB1C3==
 
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===Aim===
 
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The aim of the experiment is to perform gel electrophoresis for the amplified fragments of Pspac_oid promoter, double terminator and plasmid vector pSB1C3 and thus perform gel exraction of the required bands.
 
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===Materials and Protocol===
 
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Please refer to: [[Team:Newcastle/Gel electrophoresis| Gel electrophoresis]].
 
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===Result===
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=Preparation of subtilin immunity BioBrick primers and Phusion PCR =
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==Aims==
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'''Figure 1''': Gel electrophoresis of the ''lacI'' and Pspac_oid promoter.  
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[[Image:Newcastle_6_primer_tubes.JPG|200px|right]]
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To rehydrate the subtilin immunity primers. The six primers ordered are Primer 1 P-1, Primer 2 P-2, Primer 1 S-1, Primer 2 S-1, Primer 1 T-1 and Primer 2 T-1. The other primers are Primer 1 V-1 and Primer 2 V-1, which we already have in stock, therefore no rehydration will be needed. (Primer 1 = Forward primer and Primer 2 = Reverse primer).  
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* '''Lane 1''': 1kb DNA ladder
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Phusion PCR will then be performed, using four different DNA fragments, the plasmid vector, promotor & RBS, ''spaIFEG'' gene cluster and double terminator.
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* '''Lane 2''': Plamid pMutin4 containing Pspac_oid promoter
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* '''Lane 3''': Plamid pMutin4 containing Pspac_oid promoter
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For more information about the subtilin immunity BioBrick, please see the cloning strategy for subtilin immunity (Link)
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* '''Lane 4''': Plamid pMK-RQ (''kinA'' BioBrick) containing Pspac_oid promoter
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* '''Lane 5''': Plamid pMK-RQ (''kinA'' BioBrick) containing Pspac_oid promoter
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==Materials and Protocol==
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* '''Lane 6''': Plamid pMK-RO (stchastic switch BioBrick) containing Pspac_oid promoter
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* '''Lane 7''': Plamid pMK-RO (stchastic switch BioBrick) containing Pspac_oid promoter
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Please refer to the [[Team:Newcastle/DNA_re-hydration| DNA Re-hydration]] protocol.
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* '''Lane 8''': 100bp DNA ladder
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Please refer to the [[Team:Newcastle/PCR#Phusion_PCR| Phusion PCR]] protocol.
{|border=1
{|border=1
|-
|-
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!
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!'''Tube'''
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!'''Pspac_oid pormoter'''
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!'''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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|'''Size of the Fragment (in bp)'''
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|1
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|148 approx.
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|Plasmid Vector
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|PSB1C3
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|P1V1 forward
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|P2V1 reverse
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|53.3
 +
|2046 +
 +
|70
 +
|-
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|2
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|Promoter and RBS (pVeg-SpoVG)
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|BioBrick Bba_K143053
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|P1P1 forward
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|P2P2 reverse
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|51.7
 +
|139 +
 +
|15
 +
|-
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|3
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|''spaIFEG'' Gene Cluster
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|''B. subtilis'' ATCC 6633
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|P1S1 forward
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|P2S1 reverse
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|46.0
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|2753 +
 +
|110
 +
|-
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|4
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|Double terminator
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|pSB1AK3 consisting BBa_B0014
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|P1T1 forward
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|P2T1 reverse
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|50.9
 +
|116 +
 +
|15
|}
|}
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'''Table 3''': Table represents the size of the Pspac_oid fragment represented as bands on the gel in all the lanes.
 
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===Discussion===
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'''Table 5''': This table shows the four different Phusion PCR reactions that were carried out today. If this is successful, the four parts can be ligated together for the construction of subtilin immunity BioBrick with the help of Gibson Cloning method.
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We found two bands in the all lanes out of which one is of approximately of 150 bp is size and the other band is of 80 bp approximately in size.
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* The extension rate of the Phusion polymerase is 1 Kb/ 30 seconds. Therefore the extension time of each PCR reaction is different.
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* Note: The stock primer have to be handled with extra caution, because they will be the main stocks from which working stock solutions will be made from. Therefore, gloves have to be worn to prevent any contamination. Water will have to be from the Pure Lab Distilled Water.
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==Discussion==
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Gel electrophoresis for the PCR product will be done on[[Team:Newcastle/11_August_2010#Subtilin_Immunity_BioBrick| 11 August]].
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===Conclusion===
 
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==Gibson assembly of ''rocF'' BioBrick==
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'''Go back to our main [[Team:Newcastle/notebook| Lab book]] page'''
{{Team:Newcastle/footer}}
{{Team:Newcastle/footer}}

Latest revision as of 23:06, 27 October 2010

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Contents

Preparation of subtilin immunity BioBrick primers and Phusion PCR

Aims

Newcastle 6 primer tubes.JPG

To rehydrate the subtilin immunity primers. The six primers ordered are Primer 1 P-1, Primer 2 P-2, Primer 1 S-1, Primer 2 S-1, Primer 1 T-1 and Primer 2 T-1. The other primers are Primer 1 V-1 and Primer 2 V-1, which we already have in stock, therefore no rehydration will be needed. (Primer 1 = Forward primer and Primer 2 = Reverse primer).

Phusion PCR will then be performed, using four different DNA fragments, the plasmid vector, promotor & RBS, spaIFEG gene cluster and double terminator.

For more information about the subtilin immunity BioBrick, please see the cloning strategy for subtilin immunity (Link)

Materials and Protocol

Please refer to the DNA Re-hydration protocol.

Please refer to the Phusion PCR protocol.

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 Plasmid Vector PSB1C3 P1V1 forward P2V1 reverse 53.3 2046 + 70
2 Promoter and RBS (pVeg-SpoVG) BioBrick Bba_K143053 P1P1 forward P2P2 reverse 51.7 139 + 15
3 spaIFEG Gene Cluster B. subtilis ATCC 6633 P1S1 forward P2S1 reverse 46.0 2753 + 110
4 Double terminator pSB1AK3 consisting BBa_B0014 P1T1 forward P2T1 reverse 50.9 116 + 15

Table 5: This table shows the four different Phusion PCR reactions that were carried out today. If this is successful, the four parts can 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. Therefore the extension time of each PCR reaction is different.
  • Note: The stock primer have to be handled with extra caution, because they will be the main stocks from which working stock solutions will be made from. Therefore, gloves have to be worn to prevent any contamination. Water will have to be from the Pure Lab Distilled Water.

Discussion

Gel electrophoresis for the PCR product will be done on 11 August.


Go back to our main Lab book page

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