Team:Heidelberg/Tyrocidine week21 variation

From 2013.igem.org

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(Created page with " To design the Linker Variation Constructs, we amplified the DNA fragments LV1a, LV1b, LV2b, LV3a, LV5b and LV6 by PCR with the following protocol. ...")
 
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To design the [[Media:Construct_Table.pdf|Linker Variation Constructs]], we amplified the DNA fragments LV1a, LV1b, LV2b, LV3a, LV5b and LV6 by PCR with the following protocol.
To design the [[Media:Construct_Table.pdf|Linker Variation Constructs]], we amplified the DNA fragments LV1a, LV1b, LV2b, LV3a, LV5b and LV6 by PCR with the following protocol.
(Primer are listed on our [[Primer#Linker_Variation|primer-page]])
(Primer are listed on our [[Primer#Linker_Variation|primer-page]])
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[[File:20130919_amplification1-6.png|right|100px|thumb|Linker variation constructs 1-6 after PCR]]
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[[File:Heidelberg_20130919_amplification1-6.png|right|100px|thumb|Linker variation constructs 1-6 after PCR]]
===Results===
===Results===
We were able to amplify all of our constructs, each of them run on the expected size.
We were able to amplify all of our constructs, each of them run on the expected size.
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As we were able to amplify all af the constructs, we quantified the gained concentrations for gibson assembly.
As we were able to amplify all af the constructs, we quantified the gained concentrations for gibson assembly.
<gallery>
<gallery>
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File:20130920_amplificationbb+ind.png|Constructs of bb and indC after PCR.
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File:Heidelberg_20130920_amplificationbb+ind.png|Constructs of bb and indC after PCR.
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File:20130919_20130922_quantificationbb+ind.png|Concentrations of bb- and indC-constructs.
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File:Heidelberg_20130919_20130922_quantificationbb+ind.png|Concentrations of bb- and indC-constructs.
</gallery>
</gallery>
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Latest revision as of 17:15, 4 October 2013

To design the Linker Variation Constructs, we amplified the DNA fragments LV1a, LV1b, LV2b, LV3a, LV5b and LV6 by PCR with the following protocol. (Primer are listed on our primer-page)

What µl
primer_fw (see link above) 2
primer_rv (see link above) 2
Brevibacillus parabrevis 1
Phusion Flash 2x Master Mix 10
ddH20 5
PCR-Program LV1a, LV1b, LV2b, LV5b amplification
Cycles Temperature [°C] Time [min:s]
1 98 2:00
35 98 0:02
59,4 0:05
72 0:28
1 72 10:00
1 10 inf
PCR-Program LV3a, LV6a amplification
Cycles Temperature [°C] Time [min:s]
1 98 2:00
35 98 0:02
59,4 0:05
72 0:44
1 72 10:00
1 10 inf
Linker variation constructs 1-6 after PCR

Contents

Results

We were able to amplify all of our constructs, each of them run on the expected size.

Gel extraction

The amplified constructs LV14,LV1b,LV2b,LV3a,LV5b,LV6a were sliced out of the gel and a gel extraction performed. Afterwards, the concentration was measured with a nanodrop.

Results

Construct Concentration in ng/µl A280/A260
LV1a 41,6 1,89
LV1b 69,4 1,86
LV2b 74,4 1,97
LV3a 72,9 1,84
LV5b 56,7 1,94
LV6a 50,7 2,05

Amplification of Backbone and indC

As we want to usethe strategy we established to detect a successful transformation by the expression of indigoidine, we amplified not only the backbone, but also the indigoidine constructs. They were amplified with both, a gibson-overhang for the short-linker-constructs and the long-linker-constructs.

PCR-Program backbone short- and long-linker amplification
Cycles Temperature [°C] Time [min:s]
1 98 0:05
35 98 0:02
66,2 0:05
72 0:50
1 72 10:00
1 10 inf
PCR-Program indigoidine short- and long-linker amplification
Cycles Temperature [°C] Time [min:s]
1 98 0:05
35 98 0:02
56,1 0:05
72 1:20
1 72 10:00
1 10 inf

Results

As we were able to amplify all af the constructs, we quantified the gained concentrations for gibson assembly.

Constructs of bb and indC after PCR.

Concentrations of bb- and indC-constructs.

Construct Concentration ind ng/µl
BBs 10
BBl 40
Inds 45
Indl 45
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