Latest revision as of 23:41, 27 October 2010

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Problem

Gel electrophoresis of subtilin immunity part fragments

Aims

The aim of this experiment is to do gel extraction for the spaIFEG gene cluster amplifed on the 12th August, 2010 and the Plasmid Vector, Promoter & RBS and Double terminator amplifed on the 11th August, 2010. Finally, NanoDrop will be performed for all the fragments.

Materials and protocol

Please refer to the gel extraction and NanoDrop protocols. Instead of using the centrifuge to bind the DNA from our sample to the QIAquick column, a vacuum manifold was used instead. It is the first time it had been used. The advantage of this was that the extraction could be carried out all at the same time.

Results

Vector	Promoter	Immunity gene cluster	Double terminator
21.3 µl/ml	28.8 µl/ml	27.0 µl/ml	27.5 µl/ml

Table 1: Nanodrop spectrophotometer results. Table represents the amount of plasmid present in µl/ml quantity.

Discussion

The concentration of the fragments range from 21.3 µl/ml to 28.8 µl/ml. This is acceptable as during the gel extraction step, some of the DNA would be lost.

Conclusion

We have successfully extracted all the parts required for the Subtilin Immunity BioBrick. The Gibson cloning method will be used next week.

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@@ Line 5: / Line 5: @@
-=Subtilin Immunity BioBrick=
+=Gel electrophoresis of subtilin immunity part fragments=
 ==Aims==
-We plan to select three ''spaIFEG'' gene (part 3) PCR tubes, using Tms 51°C, 56°C and 61°C (we had confirmed it successful from yesterday's gel electrophoresis - please refer to the gel electrophoresis from [[Team:Newcastle/12_August_2010#Results| yesterday]]). Gel extraction will then be performed, along with Plasmid Vector (part 1), Promoter & RBS (part 2) and Double terminator (part 4). Finally, NanoDrop will be performed on each of these four parts.
+The aim of this experiment is to do gel extraction for the ''spaIFEG'' gene cluster amplifed on the 12th August, 2010 and the Plasmid Vector, Promoter & RBS and Double terminator amplifed on the 11th August, 2010. Finally, NanoDrop will be performed for all the fragments.
 ==Materials and protocol==
-Please refer to the [[Team:Newcastle/Gel_electrophoresis| gel electrophoresis]], [[Team:Newcastle/Gel_extraction| gel extraction]] and [[TeamNewcastleNanoDrop_Spectrophotometer| NanoDrop]] protocols. Instead of using the centrifuge to bind the DNA from our sample to the QIAquick column, a '''vacuum manifold''' was used instead. It is the first time it had been used. The advantage of this was that the extraction could be carried out much faster as we had 7 samples.
+Please refer to the [[Team:Newcastle/Gel_extraction| gel extraction]] and [[TeamNewcastleNanoDrop_Spectrophotometer| NanoDrop]] protocols. Instead of using the centrifuge to bind the DNA from our sample to the QIAquick column, a '''vacuum manifold''' was used instead. It is the first time it had been used. The advantage of this was that the extraction could be carried out all at the same time.
 [[Image:Newcastle Gel Extraction of spaIFEG genes.JPG|200px|Gel Extraction of spaIFEG genes]]
 [[Image:Newcastle Vacuum Manifold 1.JPG|150px|Setting up the tubes for the vacuum manifold]]
 [[Image:Newcastle Vacuum Manifold 2.JPG|150px|Setting up the tubes for the vacuum manifold]]
 [[Image:Newcastle Vacuum Manifold 3.JPG|200px|Vacuum manifold with the QIAquick spin columns loaded with the samples]]
-[[Image:Newcastle Vacuum Manifold 4.JPG|200px|Vacuum manifold with the QIAquick spin columns empty after the vacuum has been switched on and the samples have passed through]]
 ==Results==
-The results from the Nanodrop Spectrophotometer of the four parts are as follows:
+{|border=1
-*'''Vector''' - 21.3 ng/μl
+|-
-*'''Promoter''' - 28.8 ng/μl
+!'''Vector'''
-*'''Coding sequence''' - 27.0 ng/μl
+!'''Promoter'''
-*'''Terminator''' - 27.5 ng/μl
+!'''Immunity gene cluster'''
+!'''Double terminator'''
+|-
+|21.3 µl/ml
+|28.8 µl/ml
+|27.0 µl/ml
+|27.5 µl/ml
+|}
+'''Table 1''': Nanodrop spectrophotometer results. Table represents the amount of plasmid present in µl/ml quantity.
 ==Discussion==
-We chose the three ''spaIFEG'' gene (part 3) PCR tubes, using Tms 51°C, 56°C and 61°C because they were the mid-ranged Tms. Gel extraction was then performed, by firstly cutting the gels under U.V light, and then apply the protocol for completion.
+The concentration of the fragments range from 21.3 µl/ml to 28.8 µl/ml. This is acceptable as during the gel extraction step, some of the DNA would be lost.
 ==Conclusion==
-The results from the NanoDrop reading suggests that our results were positive, which indicates that we have successfully extracted all the parts required for the Subtilin Immunity BioBrick. Gibson cloning method will be used next week.
+We have successfully extracted all the parts required for the Subtilin Immunity BioBrick. The Gibson cloning method will be used next week.

Team:Newcastle/13 August 2010

From 2010.igem.org