Team:Newcastle/10 August 2010

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{{Team:Newcastle/mainbanner}}
{{Team:Newcastle/mainbanner}}
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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 this experiment is to perform gel electrophoresis for the amplified fragments of Pspac oid promoter, double terminator and plasmid vector pSB1C3 and then perform gel extraction 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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The experiment is divided in two separate gels. For plasmid vector pSB1C3, we used 1% agarose gel but for Psapc_oid and double terminator fragment we used 1.5% agarose gel for a better resolution.
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We used a 1Kb DNA ladder (Promega) for the gel containing plasmid vector while we used a 100bp DNA ladder (Promega) for the gel containing Pspac_oid and double terminator fragment. 
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{|border=1
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|-
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!
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!'''Pspac_oid pormoter'''
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!'''Double Terminator'''
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!'''Plasmid Vector pSB1C3'''
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|-
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|'''Size of the Fragment (in bp)'''
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|148 approx.
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|116 approx.
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|2072 approx.
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|}
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'''Table 1''': Table represents the sizes of the Pspac_oid fragment, Double terminator and plasmid vector pSB1C3 represented as bands on the gel in their respective lanes.
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===Discussion===
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We found bands  of appropriate sizes in their respective lanes.
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===Conclusion===
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Gel electrophoresis was successful and now we would be doing gel extraction of all the six fragments (including the three fragments on which we did gel electrophoresis today) fragments.
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==Gel extraction of all 6 fragments for ''rocF''==
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===Aim===
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The aim of this experiment is to perform gel extraction of the bands containing the amplified fragments of the Pspac oid promoter, double terminator and plasmid vector pSB1C3.
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===Materials and Protocol===
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Please refer to: [[Team:Newcastle/Gel extraction| Gel extraction]].
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===Result===
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The experiment is divided in two separate gels. For plasmid vector pSB1C3, we used 1% agarose gel but for Psapc_oid and double terminator fragment we used 1.5% agarose gel for a better resolution.
+
-
We used 1Kb DNA ladder (Promega) for the gel containing plasmid vector while we used 100bp DNA ladder (Promega) for the gel containing Pspac_oid and double terminator fragment. 
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{|border=1
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|-
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!
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!'''Pspac_oid pormoter'''
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!'''Double Terminator'''
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!'''Plasmid Vector pSB1C3'''
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|-
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|'''Size of the Fragment (in bp)'''
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|148 approx.
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|116 approx.
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|2072 approx.
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|}
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'''Table 2''': Table represents the sizes of the Pspac_oid fragment, Double terminator and plasmid vector pSB1C3 represented as bands on the gel in their respective lanes.
+
-
 
+
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===Discussion===
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We found bands of appropriate sizes in their respective lanes.
+
-
 
+
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===Conclusion===
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Gel electrophoresis was successful and now we would be doing gel extraction of all the six fragments (including the three fragments on which we did gel electrophoresis today) fragments.
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==Mixing the Gibson reaction buffers==
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===Aim===
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The aim of this experiment is to mix the 5X isothermal reaction buffer and 1.33X Master Mix required for Gibson DNA assembly. Please see the [[Team:Newcastle/Gibson_Cloning|Gibson cloning protocol]].
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=lacI and pVeg=
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==Aims==
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We aim to [[Team:Newcastle/Qiagen_Minipreps| extract]] pVeg and lacI from transformed ''E.coli'' DH5α which was cultured in LB broth overnight(see [[Team:Newcastle/9_August_2010|09.08.2010]])check the concentration of DNA using [[TeamNewcastleNanoDrop_Spectrophotometer| nanodrop]] and perform a [[Team:Newcastle/Restriction_digests|restriction digest]] so we can run the samples on a [[Team:Newcastle/Gel_electrophoresis| gel]] to check the insert sizes.
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==Materials and Protocols==
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*[[Team:Newcastle/Qiagen_Minipreps|Plasmid extraction protocol]]
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*[[TeamNewcastleNanoDrop_Spectrophotometer|Nanodrop protocol]]
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*[[Team:Newcastle/Restriction_digests|Restriction digest protocol]]
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{|border=1
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|Restriction digest EcoR1 and Pst1
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|-
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!DNA (in µl)
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!EcoR1 (in µl)
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!Pst1 (in µl)
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!10 times buffer (in µl)
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!Water (in µl)
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!5
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!0.5
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!0.5
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!1
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!3
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|}
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*[[Team:Newcastle/Gel_electrophoresis|Gel electrophoresis protocol]]
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*[[Team:Newcastle/Gel_extraction| Gel extraction protocol]]
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See protocols for materials
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==Results==
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===Results from Nanodrop:===
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{|border=1
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|-
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!
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!lacI 1
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!lacI 2
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!lacI 3
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!lacI 4
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!pVeg
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|-
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!Concentration of DNA ng/µl
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!122.5
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!139.4
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!130.0
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!150.0
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!155.0
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|}
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'''Table 4''': Table represents the concentrations of different amplified fragments in ng/µl.
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===Results from PCR:===
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[[Image:Newcastle_gel_10_august.jpg|500px|PCR Result]]
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{|border=1
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|-
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!Lane
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!1
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!2
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!3
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!4
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!5
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!6
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!7
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|-
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!
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!1,000bp marker
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!lacI 1
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!lacI 2
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!lacI 3
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!lacI 4
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!pVeg
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!100bp marker
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|}
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=Subtilin Immunity BioBrick=
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==Aims==
==Aims==
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Lab work today will involve the rehydration of the primers that have arrived today. 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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[[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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After the primers are ready, Phusion PCR will be performed, using four different template DNA: Plasmid Vector, Promotor & RBS, ''spaIFEG'' gene cluster and Double Terminator.
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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.
For more information about the subtilin immunity BioBrick, please see the cloning strategy for subtilin immunity (Link)
For more information about the subtilin immunity BioBrick, please see the cloning strategy for subtilin immunity (Link)
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!'''Extension time* (in seconds)'''
!'''Extension time* (in seconds)'''
|-
|-
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|align="right"| 1
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|1
|Plasmid Vector  
|Plasmid Vector  
|PSB1C3
|PSB1C3
|P1V1 forward
|P1V1 forward
|P2V1 reverse
|P2V1 reverse
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|align="right"| 53.3
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|53.3
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|align="right"| 2046 approx. or +?
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|2046 +
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|align="right"| 70
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|70
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|-
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|align="right"| 2
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|2
|Promoter and RBS (pVeg-SpoVG)
|Promoter and RBS (pVeg-SpoVG)
|BioBrick Bba_K143053
|BioBrick Bba_K143053
|P1P1 forward
|P1P1 forward
|P2P2 reverse
|P2P2 reverse
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|align="right"|51.7
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|51.7
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|align="right"|139 approx. or +?
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|139 +
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|align="right"|15
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|15
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|-
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|align="right"|3
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|3
|''spaIFEG'' Gene Cluster
|''spaIFEG'' Gene Cluster
|''B. subtilis'' ATCC 6633
|''B. subtilis'' ATCC 6633
|P1S1 forward
|P1S1 forward
|P2S1 reverse
|P2S1 reverse
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|align="right"|46.0
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|46.0
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|align="right"|2753 approx. or +?
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|2753 +
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|align="right"|110
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|110
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|-
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|align="right"|4
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|4
|Double terminator
|Double terminator
|pSB1AK3 consisting BBa_B0014
|pSB1AK3 consisting BBa_B0014
|P1T1 forward
|P1T1 forward
|P2T1 reverse
|P2T1 reverse
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|align="right"|50.9
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|50.9
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|align="right"|116 approx. or +?
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|116 +
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|align="right"|15
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|15
|}
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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.
'''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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* The extension rate of the Phusion polymerase is 1Kb/ 30 seconds. Thus the extension time of each and every PCR reaction is slightly different.
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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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* Please note: The newly arrived primer tubes have to be handled with extra caution, because they will be the main stocks which working stock solutions will be made from. Therefore, gloves have to be worn, as well as preventing any contamination. Water will be used in order to liquefy the primers and the water used will be from Pure Lab Distilled Water.
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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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==Results, Discussion and Conclusion==
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==Discussion==
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For results, discussion and conclusion, please refer to the Lab book on [[Team:Newcastle/11_August_2010#Subtilin_Immunity_BioBrick| 11 August]].
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Gel electrophoresis for the PCR product will be done on[[Team:Newcastle/11_August_2010#Subtilin_Immunity_BioBrick| 11 August]].

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.


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