Team:Paris Liliane Bettencourt/Notebook/2011/09/04

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(→To Do List)
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'''Cloning'''
'''Cloning'''
-
*The plates labelled with orange tape: "Danny's Transfos": c.f. 03/09/11
+
*The plates with the orange tape labelled: "Danny's Transfos": c.f. 03/09/11
       1) In case of failure, redo the ligations the transform.
       1) In case of failure, redo the ligations the transform.
-
       2) In case of success, S38 in S24 has to be moved into pSB1C3.  
+
       2) In case of success, all strains are waiting for a strain number.
 +
        Verification either by sequencing or PCR Colony. (This can be done after the following steps, but preferably before step 3)
 +
        S38 in S24 has to be digested, then inserted into pSB1C3.  
         The others are already in this plasmid.  
         The others are already in this plasmid.  
-
         They are to be inserted into pHM3, then transformed into subtilis.
+
         They are to be digested and inserted into pHM3.
 +
      3) All constructs inserted into pHM3 will then be transformed into subtilis.
 +
 
 +
*The plates with the orange tape labelled: "For Cyrille..."
 +
      1) In case of most probable failure, redo the transformation with the PCR tubes wrapped in orange tape.
 +
        (Box: Extra space)
 +
      2) In case of success,
 +
        Minipreps and glycerols should be made.
 +
        Minipreps should be sent for sequencing.

Revision as of 23:23, 3 September 2011

Danyel

  • Overview
  • To do list

Overview

The constructs involved in the tRNA project so far:


  • General information : all values are APPROX.
    • tRNA = 100 bp
    • T7 amber = 2.7 kb
    • GFP amber = 720 bp
    • pHyperspank = 101 bp
    • Pveg_spoVG = 120 bp
    • pT7_RBS_GFP_TT = 823 bp


  • tRNA amber suppressor
    • tRNA 
     S38: in pSB1C3
  • tRNA_TT 
     S58: in pSB1C3
  • Pveg_spoVG_tRNA 
     S38 in S24 --- [in progress]
  • Pveg_spoVG_tRNA_TT 
     S81 :  this contruct in pSB1AK3 (ampR)
     S92 :  this contruct in pHM3 (ampR/tetR) --- sequencing required and transformation in subtilis attempted by Adrien.
  • pHyperspank_tRNA 
     S38 in S82 --- [in progress]
  • pHyperspank_tRNA_TT 
     S58 in S82 --- [in progress]


  • Proteins with amber mutations
  • T7 amber_TT 
     S80 : in pSB1AK3
  • GFP amber 
     [in progress : transformation]
  • GFP_TT 
     [in progress : transformation]


  • other constructs
  • pT7_RBS_GFP_TT  : 
     S79 :  this construct in ampR plasmid (pSB1AK3?).
     *Functionality has been tested through transformation in BL21 and microscopy. (cf.27/08/11)

To Do List

in order of priority:

Sequencing

  • S92 = S81 in pHM3

Cloning

  • The plates with the orange tape labelled: "Danny's Transfos": c.f. 03/09/11 
     1) In case of failure, redo the ligations the transform.
     2) In case of success, all strains are waiting for a strain number.
        Verification either by sequencing or PCR Colony. (This can be done after the following steps, but preferably before step 3)
        S38 in S24 has to be digested, then inserted into pSB1C3. 
        The others are already in this plasmid. 
        They are to be digested and inserted into pHM3.
     3) All constructs inserted into pHM3 will then be transformed into subtilis.
  • The plates with the orange tape labelled: "For Cyrille..." 
     1) In case of most probable failure, redo the transformation with the PCR tubes wrapped in orange tape. 
       (Box: Extra space)
     2) In case of success,
        Minipreps and glycerols should be made.
        Minipreps should be sent for sequencing.
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