Team:Newcastle/7 July 2010

From 2010.igem.org

(Difference between revisions)
(Protocol)
(Chromosomal prep)
 
(14 intermediate revisions not shown)
Line 1: Line 1:
{{Team:Newcastle/mainbanner}}
{{Team:Newcastle/mainbanner}}
-
==Chromosomal prep==
+
=PCR to test chromosomal prep=
-
===Results===
+
==Aim==
-
We used Gel Electrophoresis to test whether we have the DNA we wanted. As we already know, the ''ara'' gene is about 300bps. We used hundred bp DNA ladder as a guidance for our bands. The two bands produced were from two separate ''ara'' cultures and they indeed show the bands in the same region, i.e. 300bps.  
+
To test whether the chromosome extraction from ''Bacillus subtilis'' ATCC6633 was successful.
-
[[Image:Newcastle PCR gel.jpg|400px]]
+
==Materials and Procedure==
 +
Please refer to:[[Team:Newcastle/PCR| PCR]] and [[Team:Newcastle/Gel electrophoresis| Gel electrophoresis]] for materials and protocol.
 +
We used ''ara'' forward and reverse primers for amplification.
-
===Conclusion===
+
==Results==
-
The aim of this whole experiment was to extract genomic DNA from ''B. subtilis'' strain ATCC 6633. In order to test whether we had extracted the DNA, we first used PCR to amplify and then used Gel Electrophoresis to compare the bands of ''ara'' genes to the hundred bps ladder.It worked!
+
We used gel electrophoresis to confirm the quality of our DNA. As we already know, the region our primers amplify is about 300bps. We used 100 bp DNA ladder as a guidance for our bands. The two bands produced were from two separate chromosomal preps.
-
==LacI BioBrick Construction==
+
[[Image:Newcastle_Genomic_extraction.jpg|400px]]
-
===Aims===
+
* '''Lane 1''': 100 bp DNA ladder
-
* To use PCR to extract ''lacI'' (promoter, ribosome-binding site (RBS) & coding sequence (CDS)) from plasmid pMutin4 and ligate into vector pSB1AT3 in front of red fluorescent protein (RFP).
+
* '''Lane 2''': Chromosomal DNA with ara gene 1
 +
* '''Lane 3''': Chromosomal DNA with ara gene 2
-
===Materials===
+
==Conclusion==
-
* Digested plasmids pMutin4 and pSB1AT3
+
The aim of this whole experiment was to extract genomic DNA from ''Bacillus subtilis'' ATCC 6633. In order to test whether we had extracted the DNA, we first used PCR to amplify and then used Gel Electrophoresis to compare the bands of ''ara'' genes to the 100 bp ladder. It worked!
-
* PCR product
+
-
* Restriction enzymes EcoR1 and Spe1
+
-
===Protocol===
 
-
* The digests and PCR product are run on an [[Team:Newcastle/Gel_electrophoresis|agarose gel]]
 
-
* pSB1AT3 is excised from the gel
 
-
* ''lacI'' PCR product is purified.
 
-
* Purified ''lacI'' is [[Team:Newcastle/Restriction_digests|restriction digested]] with EcoR1 and Spe1.
 
-
===Results===
 
-
* Bands were seen at the correct size for pMutin4 and pSB1AT3 and the amplified PCR product.
 
-
 
-
===Conclusion===
 
-
* The digested plasmids and the PCR product were run on an agarose gel in order to determine what was in these samples. The samples should have run at                . The samples did in fact run in this manner, and so it can be said with confidence that our protocols have worked as they should.
 
-
 
-
===Inference===
 
-
* The purified PCR product is cut to give it sticky ends complementary to the digested pSB1AT3. pSB1AT3 is excised from the gel ready for gel extraction.
 
{{Team:Newcastle/footer}}
{{Team:Newcastle/footer}}

Latest revision as of 15:09, 27 October 2010

iGEM Homepage Newcastle University BacillaFilla Homepage Image Map

Contents

PCR to test chromosomal prep

Aim

To test whether the chromosome extraction from Bacillus subtilis ATCC6633 was successful.

Materials and Procedure

Please refer to: PCR and Gel electrophoresis for materials and protocol. We used ara forward and reverse primers for amplification.

Results

We used gel electrophoresis to confirm the quality of our DNA. As we already know, the region our primers amplify is about 300bps. We used 100 bp DNA ladder as a guidance for our bands. The two bands produced were from two separate chromosomal preps.

Newcastle Genomic extraction.jpg

  • Lane 1: 100 bp DNA ladder
  • Lane 2: Chromosomal DNA with ara gene 1
  • Lane 3: Chromosomal DNA with ara gene 2

Conclusion

The aim of this whole experiment was to extract genomic DNA from Bacillus subtilis ATCC 6633. In order to test whether we had extracted the DNA, we first used PCR to amplify and then used Gel Electrophoresis to compare the bands of ara genes to the 100 bp ladder. It worked!


Newcastle University logo.png    Newcastle cbcb logo.pngNewcastle Biomedicine logo.gif    Team Newcastle CEG logo.gif
Newcastle iww logo.jpg  UNIPV Pavia Logo.gif  Newcastle BBSRC.gif    Newcastle Genevision logo.png Newcastle WelcomeTrust.jpg
FaceBook Icon