Team:DTU-Denmark/Notebook/30 June 2013

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{{:Team:DTU-Denmark/Templates/StartPage|30 June 2013}}
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=208 lab=
=208 lab=
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== Main purposes today ==
== Main purposes today ==
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New PCR with x7-polymerase.
New PCR with x7-polymerase.
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==Who was in the lab==
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==who were in the lab==
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Kristian
Kristian
==Procedure==
==Procedure==
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Made PCRs on pZA21, GFP SF TAT, GFP SF Sec and RFP all samples where made in duplicates.
Made PCRs on pZA21, GFP SF TAT, GFP SF Sec and RFP all samples where made in duplicates.
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In 19+20+21+22+23+24 RFP
In 19+20+21+22+23+24 RFP
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First program was 55°C annealing and 2:00 extension time with tube: 1+2, 3+4.
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First program was 45°C annealing and 2:00 extension time with tube: 1+2, 7+8, 13+14, 19+20.
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Second program was 64°C annealing and 2:00 extension time with tube: 5+6, 7+8.
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Second program was ramp 68°C → 60°C annealing and 2:00 extension time with tube: 3+4, 9+10, 15+16, 21+22.
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Last program was 69°C annealing and 1:00 extension time with tube: 9+10.
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Last program was a touch up program with 60°C annealing and 10 cycles with a temperature increment of 0.5°C per cycle this was looped 5 times so the final amount of annealing cycles was 50. Extension time was still 2:00 and this program was done on tube: 5+6, 11+12, 17+18, 23+24.  
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Purification was done by gel purification, see picture below.
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[[File:30.06.13 all PCR products purification gel.jpg|thumb|250px|left|Purification gel with big well number one(left uppermost) containing 1+2 next 3+4, 5+6, 7+8, 9+10, 11+12, next line from left to right; 13+17, 14+16, failed well, 21+22, 24]]
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After all PCR-products were purified the backbone was prepared for DpnI treatment and the right amount of each of the other PCR-products were mixed. Finally the DpnI treated backbone was added USER-enzyme and BSA, mixed with the appropriate PCR-products and set for reaction.
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The constructs were transformed into chemical competent ''E.coli'' right after construction and incubated 2 hours in SOC before plated on Kana plates.
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<html><p><br/><br/><br/><br/> </p></html>
==Results==
==Results==
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<hr/>
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[[File:30.06.13 all PCR products with x7 first part.jpg|thumb|left|The gel from the days PCR. From first lane is a 100 bp ladder and thereafter the well number follows the numbers of the tubes -1. Note that tube number 15 is missing]]
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[[File:30.06.13 all PCR products with x7 first part.jpg|thumb|250px|left|The gel from the days PCR. From first lane is a 100 bp ladder and thereafter the well number follows the numbers of the tubes -1. Note that tube number 15 is missing]]
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[[File:30.06.13 all PCR products with x7 second part.jpg|thumb|left|The gel from the days PCR. From first lane is a 100 bp ladder and thereafter tube 20,21,22,23 and 24. Note that there is also primer blur in subsequent wells to the right; these are just double test wells to test previous PCR products]]
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[[File:30.06.13 all PCR products with x7 second part.jpg|thumb|250px|left|The gel from the days PCR. From first lane is a 100 bp ladder and thereafter tube 20,21,22,23 and 24. Note that there is also primer blur in subsequent wells to the right; these are just double test wells to test previous PCR products]]
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<html><p><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/> </p></html>
== Conclusion from today ==
== Conclusion from today ==
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...
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All PCR-products have been made and the programs for each product have been determined.
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Navigate to the [[Team:DTU-Denmark/Notebook/29_June_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/1_July_2013|Next]] Entry
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Latest revision as of 18:09, 30 September 2013

30 June 2013

Navigate to the Previous or the Next Entry

Contents

208 lab


Main purposes today


New PCR with x7-polymerase.

Who was in the lab


Kristian

Procedure


Made PCRs on pZA21, GFP SF TAT, GFP SF Sec and RFP all samples where made in duplicates.

In tube 1+2+3+4+5+6 where pZA21. In 7+8+9+10+11+12 GFP SF TAT In 13+14+15+16+17+18 GFP SF Sec In 19+20+21+22+23+24 RFP

First program was 45°C annealing and 2:00 extension time with tube: 1+2, 7+8, 13+14, 19+20. Second program was ramp 68°C → 60°C annealing and 2:00 extension time with tube: 3+4, 9+10, 15+16, 21+22. Last program was a touch up program with 60°C annealing and 10 cycles with a temperature increment of 0.5°C per cycle this was looped 5 times so the final amount of annealing cycles was 50. Extension time was still 2:00 and this program was done on tube: 5+6, 11+12, 17+18, 23+24.

Purification was done by gel purification, see picture below.

Purification gel with big well number one(left uppermost) containing 1+2 next 3+4, 5+6, 7+8, 9+10, 11+12, next line from left to right; 13+17, 14+16, failed well, 21+22, 24


After all PCR-products were purified the backbone was prepared for DpnI treatment and the right amount of each of the other PCR-products were mixed. Finally the DpnI treated backbone was added USER-enzyme and BSA, mixed with the appropriate PCR-products and set for reaction.

The constructs were transformed into chemical competent E.coli right after construction and incubated 2 hours in SOC before plated on Kana plates.





Results


The gel from the days PCR. From first lane is a 100 bp ladder and thereafter the well number follows the numbers of the tubes -1. Note that tube number 15 is missing
The gel from the days PCR. From first lane is a 100 bp ladder and thereafter tube 20,21,22,23 and 24. Note that there is also primer blur in subsequent wells to the right; these are just double test wells to test previous PCR products

















Conclusion from today


All PCR-products have been made and the programs for each product have been determined.


Navigate to the Previous or the Next Entry