Team:DTU-Denmark/Notebook/17 July 2013

From 2013.igem.org

(Difference between revisions)
(Procedure)
 
(19 intermediate revisions not shown)
Line 1: Line 1:
-
{{:Team:DTU-Denmark/Templates/StartPage|}}
+
{{:Team:DTU-Denmark/Templates/StartPage|17 July 2013}}
-
 
+
Navigate to the [[Team:DTU-Denmark/Notebook/16_July_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/18_July_2013|Next]] Entry
-
 
+
=Lab 208=
-
=208=
+
<hr/>
<hr/>
==Main purpose==
==Main purpose==
Line 15: Line 14:
<hr/>
<hr/>
-
- miniprep of RFP in pZA21 and cycAX in pZA21  
+
* miniprep of RFP in pZA21 and cycAX in pZA21  
-
- made gycerol stocks from ON cultures supposedly containing RFP in pZA21 and cycAX in pZA21
+
* made gycerol stocks from ON cultures supposedly containing RFP in pZA21 and cycAX in pZA21
-
- gel of yesterday's PCR (to get AMO with USER endings) -> got the fragment
+
* gel of yesterday's PCR (cycAX and Nir with USER primers)
-
- purified AMO for USER from gel
+
* purified AMO for USER from gel
-
- PCR to add His-tags to construct 1 (Sec-GFP-RFP) and construct 2 (TAT-GFP-RFP)
 
-
- gel on today's PCR -> construct 1 has no product, construct 2 has an unkown product with length 900 bp
+
=== Plasmid isolation pZA21 with RFP and pZA21 with cycAX from transformants===
 +
According to standard protocol attached to GenElute™ Plasmid Miniprep Kit.
 +
 
 +
=== PCR ===
 +
 
 +
* cycAX fragment amplified with USER primers, template - isolated plasmid from colony 12 (orange transformant)
 +
We performed PCR reaction in order to check the presence of cycAX insert in pZA21 plasmid because colony PCR for this fragment showed no positive results. We based on [[Team:DTU-Denmark/Methods/PCR| standard PCR programm]] with annealing temperature of 57°C and extension time of 1:30 min.
 +
 
 +
* Nir amplified with USER primers, template - cells of ''Pseudosomonas aeruginosa'', 2 different PCR programms were used
 +
We based on [[Team:DTU-Denmark/Methods/PCR| standard PCR programm]] and used annealing temperature of 62°C and extension time of 9 min for samples 1,2 and 4 min for samples 3,4,5.
 +
 
 +
=== AMO gel purification ===
 +
PCR product was loaded on a gel in order to perform isolation from gel a fragment of desired lenght.
 +
It was performed according to procedure in Qiagen Spin miniprep kit.
 +
 
 +
 
 +
===PCR to add His-tags to Hellow World constructs===
 +
 
 +
performed PCR on the two constructs made in the Hello World project
 +
 
 +
used primers 19a, 19b for Sec-construct
 +
 
 +
used primers 20a, 20b for TAT-construct
 +
 
 +
program for Sec-construct: 60C annealing temperature and 4:00 elongation time
 +
 
 +
program for TAT-construct: 56C annealing temperature and 4:00 elongation time
 +
 
 +
 
 +
==Results==
 +
<hr/>
 +
1% agarose gel of today's PCR of the constructs with his tags.
 +
 
 +
*1: 1kb ladder
 +
*2: Sec-construct
 +
*3: Sec-construct
 +
*4: Sec-construct
 +
*5: TAT-construct
 +
*6: TAT-construct
 +
*7: TAT-construct
 +
 
 +
[[File:2013-07-17 his construct.jpg| 600px]]
 +
 
 +
== Conclusion ==
 +
<hr/>
 +
We obtained TAT construct!
 +
 
 +
Navigate to the [[Team:DTU-Denmark/Notebook/16_July_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/18_July_2013|Next]] Entry
-
- gel of colony PCR to check the inserts of last USER cloning -> empty, but the colonies that should have RFP are coloured red and one colony with supposed cycAX is coloured orange
+
{{:Team:DTU-Denmark/Templates/EndPage}}

Latest revision as of 20:41, 16 September 2013

17 July 2013

Navigate to the Previous or the Next Entry

Contents

Lab 208


Main purpose


  • Miniprep

Who was in the lab


Gosia, Henrike

Procedure


  • miniprep of RFP in pZA21 and cycAX in pZA21
  • made gycerol stocks from ON cultures supposedly containing RFP in pZA21 and cycAX in pZA21
  • gel of yesterday's PCR (cycAX and Nir with USER primers)
  • purified AMO for USER from gel


Plasmid isolation pZA21 with RFP and pZA21 with cycAX from transformants

According to standard protocol attached to GenElute™ Plasmid Miniprep Kit.

PCR

  • cycAX fragment amplified with USER primers, template - isolated plasmid from colony 12 (orange transformant)

We performed PCR reaction in order to check the presence of cycAX insert in pZA21 plasmid because colony PCR for this fragment showed no positive results. We based on standard PCR programm with annealing temperature of 57°C and extension time of 1:30 min.

  • Nir amplified with USER primers, template - cells of Pseudosomonas aeruginosa, 2 different PCR programms were used

We based on standard PCR programm and used annealing temperature of 62°C and extension time of 9 min for samples 1,2 and 4 min for samples 3,4,5.

AMO gel purification

PCR product was loaded on a gel in order to perform isolation from gel a fragment of desired lenght. It was performed according to procedure in Qiagen Spin miniprep kit.


PCR to add His-tags to Hellow World constructs

performed PCR on the two constructs made in the Hello World project

used primers 19a, 19b for Sec-construct

used primers 20a, 20b for TAT-construct

program for Sec-construct: 60C annealing temperature and 4:00 elongation time

program for TAT-construct: 56C annealing temperature and 4:00 elongation time


Results


1% agarose gel of today's PCR of the constructs with his tags.

  • 1: 1kb ladder
  • 2: Sec-construct
  • 3: Sec-construct
  • 4: Sec-construct
  • 5: TAT-construct
  • 6: TAT-construct
  • 7: TAT-construct

2013-07-17 his construct.jpg

Conclusion


We obtained TAT construct!

Navigate to the Previous or the Next Entry