Team:Nevada/Week 17

From 2012.igem.org

(Difference between revisions)
(→September 14)
Line 71: Line 71:
Jeremiah & Chris:
Jeremiah & Chris:
-
·        Develop western
+
        Develop western
-
o        No band appeared because the BL21 cells needed to be grown in Ampicillin (Amp) and Chloramphenicol (Cm) antibiotics
+
        No band appeared because the BL21 cells needed to be grown in Ampicillin (Amp) and Chloramphenicol (Cm) antibiotics
-
·        Minipre fresh expression plasmid with TBP+ from BL21 culture
+
        Minipre fresh expression plasmid with TBP+ from BL21 culture
-
o        Ran on a gel (gel #193)
+
        Ran on a gel (gel #193)
-
·        Transform plasmid into new BL21 cells
+
        Transform plasmid into new BL21 cells
-
o        Use Amp and Cm antibiotics when plating
+
        Use Amp and Cm antibiotics when plating
-
·        Prepare submission plasmid (pBP31C)
+
        Prepare submission plasmid (pBP31C)
-
o        Miniprep and digest in large quantities as to have enough for the other groups
+
        Miniprep and digest in large quantities as to have enough for the other groups
Chris and Jeremiah:
Chris and Jeremiah:
-
·        Colony PCR check 5 samples
+
      Colony PCR check 5 samples
-
o        Ran on a gel (gel #195)
+
      Ran on a gel (gel #195)

Revision as of 19:58, 26 October 2012



Protocols | Week 1 | Week 2 | Week 3 | Week 4 | Week 5 | Week 6 | Week 7 | Week 8 | Week 9 | Week 10 | Week 11 | Week 12 | Week 13 | Week 14 | Week 15 | Week 16 | Week 17 | Week 18 | Week 19


Week 17: September 10 - September 14

Contents

September 10

Michelle:
Single colony isolation of M2-BL21 from LB AMP-CM stock plate, which was followed by culturing it in 10ml LB :::AMP-CM broth and then large scale culturing with 50ml of LB AMP-CM broth.
Justin and Dafne:
Proteins were separated from cells (as done before)
Suspended in Amylose column buffer
proteins were purified using Amylose Resin High Flow (NEB)
eluted using 1M glycine: pH 10 buffer

September 11

Joe:
harvest protein from RFP-SBP
Justin and Dafne
Concentrate protein using Desalting column
Jeremiah & Chris:
Express BL21 cells by use of a L. Arabanose gradient
Took and 8 hour and overnight time sample

September 12

· Run samples on a polyacrylamide gel · Western blot protocol

September 13

Joe: Add rice to purified protein, incubate for 5 hours 

   Not enough protein - did not work

Michelle: 

   Glycerol stocked M2-BL21 before large scale culturing M2-BL21 with 50ml LB AMP-CM broth.

September 14

Joe: make 10, 10 ml cultures of RFP-SBP in Top10 cells

Michelle: 

   Transformed M2 (SBP-LRP) into BL21 cells and plated on LB AMP-CM plates.

Jeremiah & Chris: 

        Develop western
       No band appeared because the BL21 cells needed to be grown in Ampicillin (Amp) and Chloramphenicol (Cm) antibiotics
        Minipre fresh expression plasmid with TBP+ from BL21 culture
       Ran on a gel (gel #193)
        Transform plasmid into new BL21 cells
       Use Amp and Cm antibiotics when plating
        Prepare submission plasmid (pBP31C)
       Miniprep and digest in large quantities as to have enough for the other groups


Chris and Jeremiah: 

     Colony PCR check 5 samples
Ran on a gel (gel #195)

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