Boston University/Plasmid Customization

From 2007.igem.org

< Boston University(Difference between revisions)
(→Verifying the presence of the lacZ promoter in the plasmid pJQ200)
(→Overview)
 
(20 intermediate revisions not shown)
Line 11: Line 11:
{| style="width:700pt;" border="1"
{| style="width:700pt;" border="1"
|
|
-
:'''PCR amplification of lacZ'''
+
::[[Image:BU_labeled_digested_pjq200_7-19-2007.jpg]]
 +
|
 +
:::::[[Image:BU_labeled_pjq200_sacb_digestion_7-17.jpg]]
 +
|}
 +
The native sacB gene was excised from the plasmid pJQ200 with the restriction enzymes EcoRI and HindIII.  In the presence of 10% sucrose, the sacB gene acts as a suicide gene.  After ligating a gene into this multiple cloning site, the plasmid pJQ200 should no longer be sucrose sensitive and should retain the gentamicin antibiotic resistance gene.  Positive transformants will survive in the presence of gentamicin and sucrose.
Line 20: Line 24:
-
 
-
::::
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
|
 
-
:[[Image:BU_labeled_digested_pjq200_7-19-2007.jpg]]
 
-
|
 
-
:[[Image:BU_labeled_pjq200_sacb_digestion_7-17.jpg]]
 
-
|}
 
===Verifying the presence of the lacZ promoter in the plasmid pJQ200===
===Verifying the presence of the lacZ promoter in the plasmid pJQ200===
-
 
-
 
{| style="width:700pt;" border="1"
{| style="width:700pt;" border="1"
-
|
 
-
:'''Double digestion of pJQ200 with EcoRI and HindIII'''
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
::::
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
|
|
[[Image:BU_labeled_lacz_PCR_cropped_7-17.jpg]]
[[Image:BU_labeled_lacz_PCR_cropped_7-17.jpg]]
Line 73: Line 36:
[[Image:BU_labeled_lacz_PCR_7-24-2007.jpg]]
[[Image:BU_labeled_lacz_PCR_7-24-2007.jpg]]
|}
|}
 +
 +
After PCR amplification, the PCR products are run through a gel to verify the proper amplificationn of lacZ.  Positive verification means that the primers were correctly designed, the PCR was run correctly, and that lacZ exists in a plasmid in our cell cultures.
 +
 +
 +
 +
 +
 +
===PCR amplification of the global transcription factors SO1415, SO4157, hlyU===
 +
{| style="width:700pt;" border="1"
 +
|
 +
[[Image:BU_labeled_GTF_PCR_7-25-2007.jpg]]
 +
|}
 +
 +
The PCR products hlyU and SO4157 were made.  These genes will be inserted into the plasmid pJQ200 immediately downstream of the lacZ promoter that was previously inserted by our team.

Latest revision as of 15:29, 30 July 2007

Contents

Overview

Plasmid pJQ200

Excising the sacB gene from plasmid pJQ200

BU labeled digested pjq200 7-19-2007.jpg
BU labeled pjq200 sacb digestion 7-17.jpg


The native sacB gene was excised from the plasmid pJQ200 with the restriction enzymes EcoRI and HindIII. In the presence of 10% sucrose, the sacB gene acts as a suicide gene. After ligating a gene into this multiple cloning site, the plasmid pJQ200 should no longer be sucrose sensitive and should retain the gentamicin antibiotic resistance gene. Positive transformants will survive in the presence of gentamicin and sucrose.




Verifying the presence of the lacZ promoter in the plasmid pJQ200

BU labeled lacz PCR cropped 7-17.jpg

BU labeled lacz PCR 7-21-2007.jpg

BU labeled lacz PCR 7-19-2007.jpg

BU labeled lacz PCR 7-24-2007.jpg

After PCR amplification, the PCR products are run through a gel to verify the proper amplificationn of lacZ. Positive verification means that the primers were correctly designed, the PCR was run correctly, and that lacZ exists in a plasmid in our cell cultures.



PCR amplification of the global transcription factors SO1415, SO4157, hlyU

BU labeled GTF PCR 7-25-2007.jpg

The PCR products hlyU and SO4157 were made. These genes will be inserted into the plasmid pJQ200 immediately downstream of the lacZ promoter that was previously inserted by our team.

Retrieved from "http://2007.igem.org/wiki/index.php/Boston_University/Plasmid_Customization"