Difference between revisions of "Part:BBa K274004:Experience"

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When digested with BamHI and NotI, the insert did not digest as expected, only a 2kb band (backbone) and ~6kb band were visible.
 
When digested with BamHI and NotI, the insert did not digest as expected, only a 2kb band (backbone) and ~6kb band were visible.
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Agree with Team NCTU-Formosa. I wanted to clone vioC from 3-20J, 3-22F, 4-18C in 2011 distribution, all designated as Part BBa-K274004, but the sequencing results showed that they are actually vioABDE.
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Zhenrun Zhang, ''Peking_R iGEM 2011''
  
 
== '''Characterisation by 2011 iGEM NCTU_FORMOSA''' ==
 
== '''Characterisation by 2011 iGEM NCTU_FORMOSA''' ==
  
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We found out that there's point mutation in this plasmid. This mutation occurs in the Xba I restriction site which takes a lot of time for us just to digest it in order to ligase this part with our interest part.
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We found out that there's a point mutation in this plasmid. This mutation occurs in the Xba I restriction site which takes a lot of time for us just to digest it in order to ligase this part with our interest part.
  
 
The following figure shows the unsual gel electrophoresis result which the Xba I restriction site couldn't recognize the restriction site.
 
The following figure shows the unsual gel electrophoresis result which the Xba I restriction site couldn't recognize the restriction site.
  
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[[Image:DNA gel electrophoresis1.jpg]]
  
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'''Figure.1 Compare our part before and after digestion to show the point mutation of Xba I.'''
  
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We then design a primer as follows:  
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In order to correct the mutation, we then design a primer as follows:  
  
 
[[Image:Primer design 2011 NCTU.jpg]]
 
[[Image:Primer design 2011 NCTU.jpg]]
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'''Figure.2''' Design a primer to correct the point mutation
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'''Figure.2  Design a primer to correct the point mutation.'''
  
  
 
This primer include the precise sequence of the Xba I restriction site. We then do Polymerase Chain Reaction to extend the remaining sequence and amplify the exact plasmid that we want.
 
This primer include the precise sequence of the Xba I restriction site. We then do Polymerase Chain Reaction to extend the remaining sequence and amplify the exact plasmid that we want.
  
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And we also notice something that this part is actually '''vioABDE''' instead of vioABCE.  
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[[Image:DNA gel electrophoresis2.jpg]]
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'''Figure.3 Compare our part before and after digestion to show we correct the mutation successfully'''
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'''Last and the most important, we also notice something that this part is actually '''vioABDE''' instead of vioABCE.'''
  
 
In previous time, we failed in every attempts to clone out vioC from this part. We then decided to do DNA sequencing to make sure every single sequence is correct. Afterward, we found the exact sequence of vioABDE instead of vioABCE.
 
In previous time, we failed in every attempts to clone out vioC from this part. We then decided to do DNA sequencing to make sure every single sequence is correct. Afterward, we found the exact sequence of vioABDE instead of vioABCE.
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Please refer to the file for the result of sequencing :[https://static.igem.org/mediawiki/parts/d/db/037_F05_pVioABCE_Reverse.txt Sequencing rev.] and [https://static.igem.org/mediawiki/parts/2/2f/Sequencing_analysis.txt Sequencing analysis]

Latest revision as of 04:36, 5 December 2011

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K274004

User Reviews

UNIQ8380319ae6039a3c-partinfo-00000000-QINU UNIQ8380319ae6039a3c-partinfo-00000001-QINU

E.coli turned dark green after transformation of this plasmid, it is not supposed to be so since there is no promoter upstream of the operon. We have not found the reason.


Karina Arnesen, undergraduate, Alberta iGEM 2010

When digested with BamHI and NotI, the insert did not digest as expected, only a 2kb band (backbone) and ~6kb band were visible.

Agree with Team NCTU-Formosa. I wanted to clone vioC from 3-20J, 3-22F, 4-18C in 2011 distribution, all designated as Part BBa-K274004, but the sequencing results showed that they are actually vioABDE.

Zhenrun Zhang, Peking_R iGEM 2011

Characterisation by 2011 iGEM NCTU_FORMOSA

We found out that there's a point mutation in this plasmid. This mutation occurs in the Xba I restriction site which takes a lot of time for us just to digest it in order to ligase this part with our interest part.

The following figure shows the unsual gel electrophoresis result which the Xba I restriction site couldn't recognize the restriction site.

DNA gel electrophoresis1.jpg

Figure.1 Compare our part before and after digestion to show the point mutation of Xba I.


In order to correct the mutation, we then design a primer as follows:

Primer design 2011 NCTU.jpg Figure.2 Design a primer to correct the point mutation.


This primer include the precise sequence of the Xba I restriction site. We then do Polymerase Chain Reaction to extend the remaining sequence and amplify the exact plasmid that we want.

DNA gel electrophoresis2.jpg

Figure.3 Compare our part before and after digestion to show we correct the mutation successfully


Last and the most important, we also notice something that this part is actually vioABDE instead of vioABCE.

In previous time, we failed in every attempts to clone out vioC from this part. We then decided to do DNA sequencing to make sure every single sequence is correct. Afterward, we found the exact sequence of vioABDE instead of vioABCE.

Please refer to the file for the result of sequencing :Sequencing rev. and Sequencing analysis