Berkeley LBL/JoyceNotebook

From 2007.igem.org

< Berkeley LBL(Difference between revisions)
 
(6 intermediate revisions not shown)
Line 1: Line 1:
-
'''Contruction of pET3a Derivatives Containing ''bchD'' and ''bchI'''''
+
[[User:Joyxi|'''Joyce's user page]]
-
1. Amplify Rhodobacter's gene and introduce restriction sites by [[Berkeley_LBL/PCRphusion|PCR (Using Phusion Polymerase)]], which allow cloning PCR fragments into pET3a
+
[[Berkeley_LBL/JoyceRhodobacter|'''Contruction of pET3a Derivatives Containing ''bchD'' and ''bchI''''']]
-
{| border =
+
-
|-
+
-
!
+
-
!''bchD''  
+
-
!''bchI''
+
-
|-
+
[[Berkeley_LBL/JoyceCyano|'''Contruction of pET3a Derivatives Containing ''chlD'' and ''chlI''''']]
-
|length of gene fragment
+
-
|1695 b.p.
+
-
|1005 b.p.
+
-
 
+
-
|-
+
-
|Restriction sites introduced when amplified by PCR
+
-
|NdeI, BamHI
+
-
|NdeI, BglI
+
-
 
+
-
|-
+
-
|
+
-
|
+
-
|
+
-
 
+
-
 
+
-
|}
+
-
2. Do [[Berkeley_LBL/DNAGelElectrophoresis|DNA Gel Electrophoresis]] to confirm that the length of the PCR fragments are the same as the corresponding target gene fragments.
+
-
 
+
-
3. Remove any leftover PCR enzyme in the samples by [[Berkeley_LBL/PCRcleanup|PCR Clean Up/Purification]].
+
-
 
+
-
4. [[Berkeley_LBL/Digestion|Digestion for PCR Products]] using specific restriction enzymes
+
-
{| border =
+
-
|-
+
-
!''bchD''  
+
-
|NdeI, BamHI
+
-
 
+
-
|-
+
-
!''bchI''
+
-
|NdeI, BglI
+
-
|}
+
-
 
+
-
4. Digested ''bchD'' and ''bchI'' are subjected to [[Berkeley_LBL/GelExtraction|Gel Extraction]] to ensure that only pure, digested DNA is obtained before doing ligation.
+
-
 
+
-
5. Ligate digested and purified PCR fragments with digested pET3a by [[Berkeley_LBL/Ligation|Ligation]] yielding plasmids of pETBCHD and pETBCHI.
+
-
 
+
-
6. Use small portion of the plasmids to do [[Berkeley_LBL/Digestion2|Analytic Digestion]].
+
-
 
+
-
7. Do [[Berkeley_LBL/CompetentCellTransformation|KCM Competent Cell Transformation]] to subclone plasmids into DH10B competent cells, which have been done by [[Berkeley_LBL/CompetentCell|KCM Competent Cell Production]].
+
-
 
+
-
8. Pick colonies and innoculate in 2ml LB + 2ul Carbencilin in 37°C shaker overnight.
+
-
 
+
-
9. [[Berkeley_LBL/Miniprep|Miniprep]]
+
-
 
+
-
10. [[Berkeley_LBL/Digestion2|Analytic Digestion]].
+
-
 
+
-
11. [[Berkeley_LBL/Digestion|Digestion for Miniprepped DNA]]
+
-
 
+
-
12. Do [[Berkeley_LBL/CompetentCellTransformation|KCM Competent Cell Transformation]] to subclone plasmids into BL21, which have been done by [[Berkeley_LBL/CompetentCell|KCM Competent Cell Production]].
+
-
 
+
-
 
+
-
'''Protein Expression'''
+
-
 
+
-
'''Protein Analysis'''
+

Latest revision as of 02:35, 27 October 2007

Joyce's user page

Contruction of pET3a Derivatives Containing bchD and bchI

Contruction of pET3a Derivatives Containing chlD and chlI

Retrieved from "http://2007.igem.org/wiki/index.php/Berkeley_LBL/JoyceNotebook"