Team:DTU-Denmark/Notebook/13 August 2013

From 2013.igem.org

(Difference between revisions)
(lab 208)
(PCR (promoter library))
Line 22: Line 22:
*template: pZA21::RFP  
*template: pZA21::RFP  
*primers: 52a, 52b1
*primers: 52a, 52b1
 +
*annealing temp: 58.1C
 +
*elongation time: 3:00
constitutive reference
constitutive reference
*template: pZA21::RFP  
*template: pZA21::RFP  
*primers: 52a, 52b2
*primers: 52a, 52b2
 +
*annealing temp: 58.1C
 +
*elongation time: 3:00
arabinose reference
arabinose reference
-
*template:  
+
*template: pZA21::RFP with AraBAD promoter
*primers: 51a,51b1
*primers: 51a,51b1
 +
*annealing temp: 60.3C
 +
*elongation time: 4:00
program
program
Line 37: Line 43:
|  98C || 2:00 || -  
|  98C || 2:00 || -  
|-
|-
-
|  98C || 2:00 || -
+
|  98C || 0:20 || 36
|-
|-
-
98C || 2:00 || -
+
annealing temp || 1:00 || 36
|-
|-
-
98C || 2:00 || -
+
72C || extension time || 36
|-
|-
-
98C || 2:00 || -  
+
72C || 5:00 || -
 +
|-
 +
|  10C || hold || -
|-
|-
 +
|}
==Results==
==Results==

Revision as of 15:28, 13 August 2013

13 August 2013

Contents

lab 208


Main purpose


Who was in the lab


Kristian, Henrike, Gosia

Procedure


PCR (promoter library)

PCR to amplify USER fragments of the constitutive SPL, the constitutive reference promoter and the arabinose reference promoter.

Used 5% DMSO, 2 uL of mM MgCl2 and GC-buffer.

constitutive SPL

  • template: pZA21::RFP
  • primers: 52a, 52b1
  • annealing temp: 58.1C
  • elongation time: 3:00

constitutive reference

  • template: pZA21::RFP
  • primers: 52a, 52b2
  • annealing temp: 58.1C
  • elongation time: 3:00

arabinose reference

  • template: pZA21::RFP with AraBAD promoter
  • primers: 51a,51b1
  • annealing temp: 60.3C
  • elongation time: 4:00

program

temperature time cycles
98C 2:00 -
98C 0:20 36
annealing temp 1:00 36
72C extension time 36
72C 5:00 -
10C hold -

Results


Gel of SLP screening PCR products (yesterday)

Each gel is representing one row; from left to right and up to down is row A to H:

Conclusion


SLP screening PCR

The PCR run with condition E (5% DMSO, 2mM MgCl2 with GC buffer) had the highest flexibility; it yields the expected product at different annealing temperatures: 58.4 C, 59.6, C 60.6 C, 61.9 C, 63.2 C, 64.6 C, 65 C.

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