Team:DTU-Denmark/Notebook/13 August 2013

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{{:Team:DTU-Denmark/Templates/StartPage|13 August 2013}}
{{:Team:DTU-Denmark/Templates/StartPage|13 August 2013}}
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Navigate to the [[Team:DTU-Denmark/Notebook/12_August_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/14_August_2013|Next]] Entry
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=lab 208=
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=Lab 208=
<hr/>
<hr/>
==Main purpose==
==Main purpose==
<hr/>
<hr/>
 +
*PCR to amplify USER fragments of the constitutive SPL;
 +
*PCR of Nir2 with USER primers;
 +
*Gel purification of araBAD SPL.
==Who was in the lab==
==Who was in the lab==
Line 19: Line 22:
Used 5% DMSO, 2 uL of mM MgCl2 and GC-buffer.
Used 5% DMSO, 2 uL of mM MgCl2 and GC-buffer.
-
constitutive SPL
+
<u>constitutive SPL</u>
*template: pZA21::RFP  
*template: pZA21::RFP  
*primers: 52a, 52b1
*primers: 52a, 52b1
 +
*annealing temp: 58.1C
 +
*elongation time: 3:00
-
constitutive reference
+
<u>constitutive reference</u>
*template: pZA21::RFP  
*template: pZA21::RFP  
*primers: 52a, 52b2
*primers: 52a, 52b2
 +
*annealing temp: 58.1C
 +
*elongation time: 3:00
-
arabinose reference
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Wanted to make negative for the two above reactions but accidentally put the template in the master mix.
-
*template:  
+
 
 +
<u>arabinose reference</u>
 +
*template: pZA21:araBAD:RFP (labeled Ara)
*primers: 51a,51b1
*primers: 51a,51b1
 +
*annealing temp: 60.3C
 +
*elongation time: 4:00
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program
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program (used for all 3 reactions):
{| class="wikitable" style="text-align: right"
{| class="wikitable" style="text-align: right"
! temperature !! time !! cycles
! temperature !! time !! cycles
Line 37: Line 48:
|  98C || 2:00 || -  
|  98C || 2:00 || -  
|-
|-
-
|  98C || 2:00 || -
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|  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 || -
 +
|-
 +
|}
 +
 +
===PCR of Nir2 with USER primers===
 +
 +
 +
{| class="wikitable" style="text-align: right"
 +
! sample nr. !! buffer !! %DMSO !! program
 +
|-
 +
|  1 || GC || 2 || 55C
 +
|-
 +
|  2 ||GC || 3 || 55C
 +
|-
 +
|  3 || GC || 5 || 55C
 +
|-
 +
|  4 ||  HF|| 2 || 55C
 +
|-
 +
|  5 || HF || 3 || 55C
 +
|-
 +
|  6 || HF || 5 || 55C
 +
|-
 +
|  7 || GC || 2 || ramp
 +
|-
 +
|  8 || GC || 3 || ramp
 +
|-
 +
|  9 || GC || 5 || ramp
 +
|-
 +
|  10 || HF || 2 || ramp
 +
|-
 +
|  11 || HF || 3 || ramp
 +
|-
 +
|  12 || HF || 5 || ramp
 +
|-
 +
|}
 +
 +
* PCR mix according to standard protocol with changes: addition of DMSO in 3 different final concentrations (2%, 3%, 5%); two different buffers (HF 5x and GC 5x), amount of added water was dependent on volume of added DMSO.
 +
* Primers for Nir2 - 40a, 40b
 +
* Template - fragment Nir2 amplified with non-uracil primers, 3uL were used
 +
* Polymerase x7
 +
* Program A99, samles 1-6, was based on standard PCR program with 55 C and 1 min of annealing parameters and 5 min of extension time.
 +
* Program A1, samples 7-12, PCR with initial annealing temperature of 59C falling down to 53C (rate 0.1 C/min). 5 min of extension time.
 +
 +
===Gel purification===
 +
 +
Gel purified araBAD SPL from yesterdays screening PCR
==Results==
==Results==
<hr/>
<hr/>
-
Each gel is representing one row [[Team:DTU-Denmark/Notebook/12_August_2013#Screening_PCR_on_pZA21:araBAD:RFP_spl| 17-07-2013]]; from left to right and up to down is row A to H:
+
===Gel of SLP screening PCR products (yesterday)===
 +
Each gel is representing one [[Team:DTU-Denmark/Notebook/12_August_2013#Screening_PCR_on_pZA21:araBAD:RFP_spl| row]]; from left to right and up to down is row A to H:
<gallery>
<gallery>
Line 60: Line 118:
File:Row H.png
File:Row H.png
</gallery>
</gallery>
 +
 +
===Gel of todays PCR reactions===
 +
 +
* 1kb ladder
 +
* Nir2 PCR - sample 1
 +
* Nir2 PCR - sample 2
 +
* Nir2 PCR - sample 3
 +
* Nir2 PCR - sample 4
 +
* Nir2 PCR - sample 5
 +
* Nir2 PCR - sample 6
 +
* Nir2 PCR - sample 7
 +
* Nir2 PCR - sample 8
 +
* Nir2 PCR - sample 9
 +
* Nir2 PCR - sample 10
 +
* Nir2 PCR - sample 11
 +
* Nir2 PCR - sample 12
 +
* constitutive SPL 1
 +
* constitutive SPL 2 (duplicate)
 +
* constitutive promoter reference 1
 +
* constitutive promoter reference 2 (duplicate)
 +
* constitutive promoter reference 3 (very small sample, there was almost nothing left in the master mix)
 +
* 1kb ladder
 +
 +
[[File:2013-08-13 nir2 const spl ref.jpg|600px]]
==Conclusion==
==Conclusion==
<hr/>
<hr/>
-
 
+
===SLP screening PCR===
 +
The PCR run with condition E (5% DMSO, 2mM MgCl<sub>2</sub> 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.
Navigate to the [[Team:DTU-Denmark/Notebook/12_August_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/14_August_2013|Next]] Entry
Navigate to the [[Team:DTU-Denmark/Notebook/12_August_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/14_August_2013|Next]] Entry
{{:Team:DTU-Denmark/Templates/EndPage}}
{{:Team:DTU-Denmark/Templates/EndPage}}

Latest revision as of 11:57, 4 October 2013

13 August 2013

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Contents

Lab 208

Main purpose

  • PCR to amplify USER fragments of the constitutive SPL;
  • PCR of Nir2 with USER primers;
  • Gel purification of araBAD SPL.

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

Wanted to make negative for the two above reactions but accidentally put the template in the master mix.

arabinose reference

  • template: pZA21:araBAD:RFP (labeled Ara)
  • primers: 51a,51b1
  • annealing temp: 60.3C
  • elongation time: 4:00

program (used for all 3 reactions):

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

PCR of Nir2 with USER primers

sample nr. buffer  %DMSO program
1 GC 2 55C
2 GC 3 55C
3 GC 5 55C
4 HF 2 55C
5 HF 3 55C
6 HF 5 55C
7 GC 2 ramp
8 GC 3 ramp
9 GC 5 ramp
10 HF 2 ramp
11 HF 3 ramp
12 HF 5 ramp
  • PCR mix according to standard protocol with changes: addition of DMSO in 3 different final concentrations (2%, 3%, 5%); two different buffers (HF 5x and GC 5x), amount of added water was dependent on volume of added DMSO.
  • Primers for Nir2 - 40a, 40b
  • Template - fragment Nir2 amplified with non-uracil primers, 3uL were used
  • Polymerase x7
  • Program A99, samles 1-6, was based on standard PCR program with 55 C and 1 min of annealing parameters and 5 min of extension time.
  • Program A1, samples 7-12, PCR with initial annealing temperature of 59C falling down to 53C (rate 0.1 C/min). 5 min of extension time.

Gel purification

Gel purified araBAD SPL from yesterdays screening PCR

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:

Row A.png
Row B.png
Row C.png
Row D.png
Row E.png
Row F.png
Row G.png
Row H.png

Gel of todays PCR reactions

  • 1kb ladder
  • Nir2 PCR - sample 1
  • Nir2 PCR - sample 2
  • Nir2 PCR - sample 3
  • Nir2 PCR - sample 4
  • Nir2 PCR - sample 5
  • Nir2 PCR - sample 6
  • Nir2 PCR - sample 7
  • Nir2 PCR - sample 8
  • Nir2 PCR - sample 9
  • Nir2 PCR - sample 10
  • Nir2 PCR - sample 11
  • Nir2 PCR - sample 12
  • constitutive SPL 1
  • constitutive SPL 2 (duplicate)
  • constitutive promoter reference 1
  • constitutive promoter reference 2 (duplicate)
  • constitutive promoter reference 3 (very small sample, there was almost nothing left in the master mix)
  • 1kb ladder

2013-08-13 nir2 const spl ref.jpg

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.

Navigate to the Previous or the Next Entry

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