Team:DTU-Denmark/Notebook/4 September 2013
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(→Colony PCR to confirm AMO and cycAX inserts in our Biobricks) |
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+ | ===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. | ||
==Results== | ==Results== |
Revision as of 11:46, 6 September 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
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.
Results
Conclusion
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