Team:DTU-Denmark/Notebook/4 September 2013

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{{:Team:DTU-Denmark/Templates/StartPage|4 September 2013}}
{{:Team:DTU-Denmark/Templates/StartPage|4 September 2013}}
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=Lab 208=
=Lab 208=
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==Main purpose==
==Main purpose==
<hr/>
<hr/>
-
 
+
*Colony PCR to confirm AMO and cycAX inserts in our Biobricks
==Who was in the lab==
==Who was in the lab==
<hr/>
<hr/>
 +
Henrike, Kristian
==Procedure==
==Procedure==
<hr/>
<hr/>
 +
===Colony PCR to confirm AMO and cycAX inserts in our Biobricks===
-
==Results==
+
Used Q5 master mix, 50 uL.
-
==Conclusion==
+
{| class="wikitable" style="text-align: right"
-
<hr/>
+
! compound !! volume (uL)
 +
|-
 +
| Q5 || 25
 +
|-
 +
| FW || 3
 +
|-
 +
| RV || 3
 +
|-
 +
| template || 1
 +
|-
 +
| MQ || 18
 +
|-
 +
|}
 +
 
 +
Following primer pairs:
 +
* FW_2_AMO_Seq, RV_3_AMO_Seq, - 63C - expected fragment length: 650bp
 +
* FW_1_cyc_Seq, RV_2_cyc_Seq - 61C - expected fragment length: 649bp
 +
 
 +
Program:
 +
{| class="wikitable" style="text-align: right"
 +
! temperature !! time !! cycles
 +
|-
 +
|  98C || 10:00 || -
 +
|-
 +
|  98C || 0:10 || 35
 +
|-
 +
|  annealing temperature || 0:30 || 35
 +
|-
 +
|  72C || 0:30 || 35
 +
|-
 +
|  72C || 5:00 || -
 +
|-
 +
|  10C || hold || -
 +
|-
 +
|}
 +
 
 +
===PCR to amplify HAO extraction fragment and HAO USER fragment===
 +
 
 +
The last attempts to amplify HAO with USER endings have failed. Maybe the template (HAO extraction fragment) is of poor quality, so we will try to amplify and purify this.
 +
 
 +
Reaction mix: Standard
 +
 
 +
primers: 8a, 8b
 +
 
 +
template: N. europeae culture (both from -20 and -80)
 +
 
 +
Ran on two blocks with two different annealing temperatures and two different templates, so 4 samples in total.
 +
 
 +
program:
 +
{| class="wikitable" style="text-align: right"
 +
! temperature !! time !! cycles
 +
|-
 +
|  98C || 10:00 || -
 +
|-
 +
|  98C || 0:20 || 35
 +
|-
 +
|  57C/59C || 0:30 || 35
 +
|-
 +
|  72C || 3:00 || 35
 +
|-
 +
|  72C || 5:00 || -
 +
|-
 +
|  10C || hold || -
 +
|-
 +
|}
 +
 
 +
Since this had to be ran we also made 4 new reactions to amplify HAO User from the old template. Used touchdown(62C -> 55C) on the Eppendorf and the two blocks running on the old thermocycler. For details see the sample annotations on the gel tomorrow.

Latest revision as of 10:52, 4 October 2013

4 September 2013

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Contents

Lab 208

Main purpose

  • Colony PCR to confirm AMO and cycAX inserts in our Biobricks

Who was in the lab

Henrike, Kristian

Procedure

Colony PCR to confirm AMO and cycAX inserts in our Biobricks

Used Q5 master mix, 50 uL.

compound volume (uL)
Q5 25
FW 3
RV 3
template 1
MQ 18

Following primer pairs:

  • FW_2_AMO_Seq, RV_3_AMO_Seq, - 63C - expected fragment length: 650bp
  • FW_1_cyc_Seq, RV_2_cyc_Seq - 61C - expected fragment length: 649bp

Program:

temperature time cycles
98C 10:00 -
98C 0:10 35
annealing temperature 0:30 35
72C 0:30 35
72C 5:00 -
10C hold -

PCR to amplify HAO extraction fragment and HAO USER fragment

The last attempts to amplify HAO with USER endings have failed. Maybe the template (HAO extraction fragment) is of poor quality, so we will try to amplify and purify this.

Reaction mix: Standard

primers: 8a, 8b

template: N. europeae culture (both from -20 and -80)

Ran on two blocks with two different annealing temperatures and two different templates, so 4 samples in total.

program:

temperature time cycles
98C 10:00 -
98C 0:20 35
57C/59C 0:30 35
72C 3:00 35
72C 5:00 -
10C hold -

Since this had to be ran we also made 4 new reactions to amplify HAO User from the old template. Used touchdown(62C -> 55C) on the Eppendorf and the two blocks running on the old thermocycler. For details see the sample annotations on the gel tomorrow.


Navigate to the Previous or the Next Entry

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