Team:DTU-Denmark/Notebook/27 August 2013

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(Difference between revisions)
(→PCR reactions)
(→PCR reactions)
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===PCR reactions===
===PCR reactions===
 +
 +
Set up PCR reactions for:
 +
* Biobrick backbone pSB1C3 with USER endings fitting for our inserts
 +
* constitutive reference promoter in pZA21 (for SPL)
 +
* pZA21::ara vector with USER endings fitting for Nir1 and Nir2 inserts
 +
 +
<u>first round of PCRs</u>:
 +
 +
pSB1C3:
 +
{| class="wikitable" style="text-align: right"
 +
! temperature !! time !! cycles
 +
|-
 +
|  98C || 2:00 || -
 +
|-
 +
|  98C || 0:10 || 36
 +
|-
 +
|  70C || 0:45 || 36
 +
|-
 +
|  72C || 1:30 || 36
 +
|-
 +
|  72C || 5:00 || -
 +
|-
 +
|  10C || hold || -
 +
|-
 +
|}
 +
 +
pZA21::ara for Nir:
 +
{| class="wikitable" style="text-align: right"
 +
! temperature !! time !! cycles
 +
|-
 +
|  98C || 2:00 || -
 +
|-
 +
|  98C || 0:20 || 36
 +
|-
 +
|  60C || 1:00 || 36
 +
|-
 +
|  72C || 2:00 || 36
 +
|-
 +
|  72C || 5:00 || -
 +
|-
 +
|  10C || hold || -
 +
|-
 +
|}
 +
 +
constitutive reference promoter:
 +
{| class="wikitable" style="text-align: right"
 +
! temperature !! time !! cycles
 +
|-
 +
|  98C || 2:00 || -
 +
|-
 +
|  98C || 0:20 || 36
 +
|-
 +
|  58.1C || 1:00 || 36
 +
|-
 +
|  72C || 3:00 || 36
 +
|-
 +
|  72C || 5:00 || -
 +
|-
 +
|  10C || hold || -
 +
|-
 +
|}
 +
{| class="wikitable" style="text-align: right"
{| class="wikitable" style="text-align: right"
Line 42: Line 104:
program:
program:
-
{| class="wikitable" style="text-align: right"
 
-
! temperature !! time !! cycles
 
-
|-
 
-
|  98C || 10:00 || -
 
-
|-
 
-
|  98C || 0:10 || 36
 
-
|-
 
-
|  annealing temperature || 0:30 || 36
 
-
|-
 
-
|  72C || 0:20 || 36
 
-
|-
 
-
|  72C || 5:00 || -
 
-
|-
 
-
|  10C || hold || -
 
-
|-
 
-
|}
 
==Results==
==Results==

Revision as of 17:55, 27 August 2013

27 August 2013

Contents

lab 208

Main purpose

Who was in the lab

Procedure

PCR reactions

Set up PCR reactions for:

  • Biobrick backbone pSB1C3 with USER endings fitting for our inserts
  • constitutive reference promoter in pZA21 (for SPL)
  • pZA21::ara vector with USER endings fitting for Nir1 and Nir2 inserts

first round of PCRs:

pSB1C3:

temperature time cycles
98C 2:00 -
98C 0:10 36
70C 0:45 36
72C 1:30 36
72C 5:00 -
10C hold -

pZA21::ara for Nir:

temperature time cycles
98C 2:00 -
98C 0:20 36
60C 1:00 36
72C 2:00 36
72C 5:00 -
10C hold -

constitutive reference promoter:

temperature time cycles
98C 2:00 -
98C 0:20 36
58.1C 1:00 36
72C 3:00 36
72C 5:00 -
10C hold -


compound amount (in uL)
standard mix additive mix
dNTPs 1 1
X7 ploymerase 0.5 0.5
HF buffer 10 10
MilliQ 31.5 27
FW primer 3 3
RV primer 3 3
template 1 1
DMSO - 2.5
50 mM MgCl2 - 2

Template was made by resuspending 1 culture in 100uL MilliQ.

program:

Results

Gel

  • 1 kb ladder
  • pSB1C3 for Biobricks, HF buffer
  • pSB1C3 for Biobricks, HF buffer, 5%DMSO
  • pSB1C3 for Biobricks, GC buffer
  • pSB1C3 for Biobricks, GC buffer, 5%DMSO
  • negative pSB1C3 for Biobricks
  • pZA21::ara for USER ligation with Nir
  • pZA21::ara for USER ligation with Nir (duplicate)
  • 1 kb ladder

Note: It seems the ladder overflew.

2013-08-27 bb pzaarafornir.jpg

Conclusion

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