The aim of this experiment is to assemble the fragments we extracted [[Team:Newcastle/10_August_2010|yesterday]] in order to construct the [[Team:Newcastle/Urease|''rocF'' BioBrick]] and clone it into pSB1C3 in a single step isothermal reaction.
-
==Materials and Protocol==
-
Please refer to: [[Team:Newcastle/Gibson Cloning|Gibson cloning]] for materials required and the protocol for the reaction.
-
==Result==
-
The final result about the success of the Gibson reaction will be found on 13th August, 2010.
-
==Discussion==
-
We followed the whole procedure perfectly and later today after the reaction is complete, we would be transforming ''E. coli'' DH5α cells. We would be setting up 48 hours culture and by 13th August, 2010 we would be getting the result about the ligation of all 6 fragments of ''rocF''.
-
-
==Conclusion==
-
The procedure went successfully and the result will be out by the end of 13th August, 2010.
-
-
=Transformation of ''E. coli'' DH5α cells with ligated ''rocF'' fragments=
-
==Aim==
-
The aim of this experiment is to transform ''E. coli'' DH5α cells with the ligated fragments (by Gibson menthod) of ''rocF'' BioBrick so as to create multiple copies of the ligated fragments.
-
-
==Materials and Protocol==
-
Please refer to: [[Team:Newcastle/Transformation of E. coli|Transformation of ''E. coli'']].
-
-
After transformation, 15 μl, 50 μl and 100 μl of transformed ''E. coli'' were plated onto 1.5% agar plate containing chloramphenicol as a selection marker.
-
-
==Result==
-
We would be putting the plates at 37°C for 48 hours and on 13th August, 2010, we would be searching for the colonies present on the plates.
-
-
==Discussion==
-
If the Gibson reaction has worked perfectly and if the transformation went successfully, then we would be having colonies on the agar plates.
-
-
==Conclusion==
-
The procedure went successfully and the result will be out by the end of 13th August, 2010.
-
=Subtilin Immunity BioBrick=
+
=Gel electrophoresis for the subtilin immunity fragments=
==Aims==
==Aims==
-
Our aims for today are to run the gel electrophoresis to check whether we have the correct fragement sizes on the four parts that are we amplified [[Team:Newcastle/10 August 2010#Subtilin_Immunity_BioBrick| yesterday]]. If the PCR worked, we will then perform gel extraction and then perform another gel electrophoresis for the extracted gel in order to obtain our BioBrick parts.
+
The aim of the experiment is to check whether we have the correct fragment sizes on the four subtilin fragments that are we have amplified [[Team:Newcastle/10 August 2010#Subtilin_Immunity_BioBrick| yesterday]].
The correct band size were observed for for the Plasmid Vector (lane 2), Promoter & RBS (lane 3) and Double terminator (lane 5). However no band was observed for the ''spaIFEG''(lane 4).
-
''spaIFEG'' PCR tube (lane 4) did not show up. We think that this occurrence was due to the Tm error (it should be 63°C as opposed to 46°C). Therefore, another PCR and gel electrophoresis were performed. Please see [[Team:Newcastle/12_August_2010| tomorrow's]] lab book page for this.
+
-
+
-
=Transformation of hyperspank and spoVG=
+
-
+
-
==Aim==
+
-
+
-
To transform competent ''E. coli'' DH5α with hyperspank and spoVG.
+
-
+
-
==Materials and Protocol==
+
-
+
-
Please refer to [[TeamNewcastleTransformation_of_E._coli|transformation of ''E. coli'']].
+
+
==Conclusion==
+
No band was observed for the ''spaIFEG''. This could be due to the wrong melting temperature.
'''Go back to our main [[Team:Newcastle/notebook| Lab book]] page'''
'''Go back to our main [[Team:Newcastle/notebook| Lab book]] page'''
Figure 1: Gel electrophoresis of the PCR products of the parts required for the subtilin immunity BioBrick.
Lane 1: 1 kb DNA ladder
Lane 2: Plasmid Vector (pSB1C3)
Lane 3: Promoter and RBS (pVeg-SpoVG)
Lane 4: spaIFEG Gene Cluster
Lane 5: Double terminator
Lane 6: 100 bp DNA ladder
Discussion
The correct band size were observed for for the Plasmid Vector (lane 2), Promoter & RBS (lane 3) and Double terminator (lane 5). However no band was observed for the spaIFEG(lane 4).
Conclusion
No band was observed for the spaIFEG. This could be due to the wrong melting temperature.