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Revision as of 08:39, 4 October 2013

Delftibactin. Bringing it all together.

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Week 1

This week the project DelRest was launched. In order to clone the Delftibactin cluster from D. Acidovorans into E.coli we decided to use Gibson cloning. Therefore Gibson primers were designed to amplify our target backbone pSB4K5 with an overlap to DelA. Furthermore the Gibson primer connecting DelOP and DelAG introduces the ribosome binding site BBa_BNILS. Accordingly the Gibson primer for joining DelL with DelOP includes the ribosome binding site BBa_BNILS. The very last gibson primer used to amplify DelL consequently has an overlap to the beginning of the mRFP reporter gene connected to pSB4K5. Check out our vectormap if you are curious about the detailed primer design. The goal of this week was to amplify our previously assembled backbone with the intended overlaps as well as the desired genes from our host organism D. Acidovorans .

Week 2

This week the project DelRest was launched. In order to clone the Delftibactin cluster from D. Acidovorans into E.coli we decided to use Gibson cloning. Therefore Gibson primers were designed to amplify our target backbone pSB4K5 with an overlap to DelA. Furthermore the Gibson primer connecting DelOP and DelAG introduces the ribosome binding site BBa_BNILS. Accordingly the Gibson primer for joining DelL with DelOP includes the ribosome binding site BBa_BNILS. The very last gibson primer used to amplify DelL consequently has an overlap to the beginning of the mRFP reporter gene connected to pSB4K5. Check out our vectormap if you are curious about the detailed primer design. The goal of this week was to amplify our previously assembled backbone with the intended overlaps as well as the desired genes from our host organism D. Acidovorans .

Week 3

This week the project DelRest was launched. In order to clone the Delftibactin cluster from D. Acidovorans into E.coli we decided to use Gibson cloning. Therefore Gibson primers were designed to amplify our target backbone pSB4K5 with an overlap to DelA. Furthermore the Gibson primer connecting DelOP and DelAG introduces the ribosome binding site BBa_BNILS. Accordingly the Gibson primer for joining DelL with DelOP includes the ribosome binding site BBa_BNILS. The very last gibson primer used to amplify DelL consequently has an overlap to the beginning of the mRFP reporter gene connected to pSB4K5. Check out our vectormap if you are curious about the detailed primer design. The goal of this week was to amplify our previously assembled backbone with the intended overlaps as well as the desired genes from our host organism D. Acidovorans .

Week 4

This week the project DelRest was launched. In order to clone the Delftibactin cluster from D. Acidovorans into E.coli we decided to use Gibson cloning. Therefore Gibson primers were designed to amplify our target backbone pSB4K5 with an overlap to DelA. Furthermore the Gibson primer connecting DelOP and DelAG introduces the ribosome binding site BBa_BNILS. Accordingly the Gibson primer for joining DelL with DelOP includes the ribosome binding site BBa_BNILS. The very last gibson primer used to amplify DelL consequently has an overlap to the beginning of the mRFP reporter gene connected to pSB4K5. Check out our vectormap if you are curious about the detailed primer design. The goal of this week was to amplify our previously assembled backbone with the intended overlaps as well as the desired genes from our host organism D. Acidovorans .

Week 5

This week the project DelRest was launched. In order to clone the Delftibactin cluster from D. Acidovorans into E.coli we decided to use Gibson cloning. Therefore Gibson primers were designed to amplify our target backbone pSB4K5 with an overlap to DelA. Furthermore the Gibson primer connecting DelOP and DelAG introduces the ribosome binding site BBa_BNILS. Accordingly the Gibson primer for joining DelL with DelOP includes the ribosome binding site BBa_BNILS. The very last gibson primer used to amplify DelL consequently has an overlap to the beginning of the mRFP reporter gene connected to pSB4K5. Check out our vectormap if you are curious about the detailed primer design. The goal of this week was to amplify our previously assembled backbone with the intended overlaps as well as the desired genes from our host organism D. Acidovorans .

Week 6

This week the project DelRest was launched. In order to clone the Delftibactin cluster from D. Acidovorans into E.coli we decided to use Gibson cloning. Therefore Gibson primers were designed to amplify our target backbone pSB4K5 with an overlap to DelA. Furthermore the Gibson primer connecting DelOP and DelAG introduces the ribosome binding site BBa_BNILS. Accordingly the Gibson primer for joining DelL with DelOP includes the ribosome binding site BBa_BNILS. The very last gibson primer used to amplify DelL consequently has an overlap to the beginning of the mRFP reporter gene connected to pSB4K5. Check out our vectormap if you are curious about the detailed primer design. The goal of this week was to amplify our previously assembled backbone with the intended overlaps as well as the desired genes from our host organism D. Acidovorans .

Week 7

This week the project DelRest was launched. In order to clone the Delftibactin cluster from D. Acidovorans into E.coli we decided to use Gibson cloning. Therefore Gibson primers were designed to amplify our target backbone pSB4K5 with an overlap to DelA. Furthermore the Gibson primer connecting DelOP and DelAG introduces the ribosome binding site BBa_BNILS. Accordingly the Gibson primer for joining DelL with DelOP includes the ribosome binding site BBa_BNILS. The very last gibson primer used to amplify DelL consequently has an overlap to the beginning of the mRFP reporter gene connected to pSB4K5. Check out our vectormap if you are curious about the detailed primer design. The goal of this week was to amplify our previously assembled backbone with the intended overlaps as well as the desired genes from our host organism D. Acidovorans .

Week 8

This week the project DelRest was launched. In order to clone the Delftibactin cluster from D. Acidovorans into E.coli we decided to use Gibson cloning. Therefore Gibson primers were designed to amplify our target backbone pSB4K5 with an overlap to DelA. Furthermore the Gibson primer connecting DelOP and DelAG introduces the ribosome binding site BBa_BNILS. Accordingly the Gibson primer for joining DelL with DelOP includes the ribosome binding site BBa_BNILS. The very last gibson primer used to amplify DelL consequently has an overlap to the beginning of the mRFP reporter gene connected to pSB4K5. Check out our vectormap if you are curious about the detailed primer design. The goal of this week was to amplify our previously assembled backbone with the intended overlaps as well as the desired genes from our host organism D. Acidovorans .

Week 9

This week the project DelRest was launched. In order to clone the Delftibactin cluster from D. Acidovorans into E.coli we decided to use Gibson cloning. Therefore Gibson primers were designed to amplify our target backbone pSB4K5 with an overlap to DelA. Furthermore the Gibson primer connecting DelOP and DelAG introduces the ribosome binding site BBa_BNILS. Accordingly the Gibson primer for joining DelL with DelOP includes the ribosome binding site BBa_BNILS. The very last gibson primer used to amplify DelL consequently has an overlap to the beginning of the mRFP reporter gene connected to pSB4K5. Check out our vectormap if you are curious about the detailed primer design. The goal of this week was to amplify our previously assembled backbone with the intended overlaps as well as the desired genes from our host organism D. Acidovorans .

Week 10

This week the project DelRest was launched. In order to clone the Delftibactin cluster from D. Acidovorans into E.coli we decided to use Gibson cloning. Therefore Gibson primers were designed to amplify our target backbone pSB4K5 with an overlap to DelA. Furthermore the Gibson primer connecting DelOP and DelAG introduces the ribosome binding site BBa_BNILS. Accordingly the Gibson primer for joining DelL with DelOP includes the ribosome binding site BBa_BNILS. The very last gibson primer used to amplify DelL consequently has an overlap to the beginning of the mRFP reporter gene connected to pSB4K5. Check out our vectormap if you are curious about the detailed primer design. The goal of this week was to amplify our previously assembled backbone with the intended overlaps as well as the desired genes from our host organism D. Acidovorans .

Week 11

Cras justo odio, dapibus ac facilisis in, egestas eget quam. Donec id elit non mi porta gravida at eget metus. Nullam id dolor id nibh ultricies vehicula ut id elit.

Learn more

At the beginning of the week, we could verify that the Gibson Assembly for Tripeptide I was indeed positive, however, the other Gibson Assemblies did not work properly. Instead of picking new colonies, we decided to optimize the Gibson recipe instead, as backbone religations were the most common problem. With these improved protocols, we used Gibson Assembly for the Dipeptide, Tripeptide II and Tetrapeptide I, later that week, Tetrapeptide II followed. After the Transformation to DH10β cells and screening by restriction digest we could send samples for the Dipeptide and Tetrapeptide I to sequencing and obtained a positive alignment. Hence we transformed BAP I cells with the positive constructs. The same was...

Week 12

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Browse gallery

At the beginning of the week, we could verify that the Gibson Assembly for Tripeptide I was indeed positive, however, the other Gibson Assemblies did not work properly. Instead of picking new colonies, we decided to optimize the Gibson recipe instead, as backbone religations were the most common problem. With these improved protocols, we used Gibson Assembly for the Dipeptide, Tripeptide II and Tetrapeptide I, later that week, Tetrapeptide II followed. After the Transformation to DH10β cells and screening by restriction digest we could send samples for the Dipeptide and Tetrapeptide I to sequencing and obtained a positive alignment. Hence we transformed BAP I cells with the positive constructs. The same was...

Week 13

Cras justo odio, dapibus ac facilisis in, egestas eget quam. Donec id elit non mi porta gravida at eget metus. Nullam id dolor id nibh ultricies vehicula ut id elit.

Browse gallery

Week 14

At the beginning of the week, we could verify that the Gibson Assembly for Tripeptide I was indeed positive, however, the other Gibson Assemblies did not work properly. Instead of picking new colonies, we decided to optimize the Gibson recipe instead, as backbone religations were the most common problem. With these improved protocols, we used Gibson Assembly for the Dipeptide, Tripeptide II and Tetrapeptide I, later that week, Tetrapeptide II followed.

Week 15

Cras justo odio, dapibus ac facilisis in, egestas eget quam. Donec id elit non mi porta gravida at eget metus. Nullam id dolor id nibh ultricies vehicula ut id elit.

Learn more

Week 16

Cras justo odio, dapibus ac facilisis in, egestas eget quam. Donec id elit non mi porta gravida at eget metus. Nullam id dolor id nibh ultricies vehicula ut id elit.

Browse gallery

Week 17

Cras justo odio, dapibus ac facilisis in, egestas eget quam. Donec id elit non mi porta gravida at eget metus. Nullam id dolor id nibh ultricies vehicula ut id elit.

Browse gallery

Week 18

dumm dummy

Week 19

After successful dumm dummy

Week 20

dumm dummy

Week 21

dummy

Week 22

dummmmmy

Week 23

Cras justo odio, dapibus ac facilisis in, egestas eget quam. Donec id elit non mi porta gravida at eget metus. Nullam id dolor id nibh ultricies vehicula ut id elit.

Browse gallery

Methods:

Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet.Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. .

06-07-2013

Amplification from FS_02 to FS_03; 5.3 kb

PCR for amplification of del fragments E-G and A-E, AE+=PCR AE with DMSO, AE-=PCR without DMSO, M= Marker; wanted amplicon sizes are: AE=5.3kb, EG=11.0

Reaction

what	µL
D. acidovorans DSM-39	1
FS_02: (1/10)	1
FS_03: (1/10)	1
Phusion Master Mix	10
DMSO	1/-
dd H₂O	6/7

PCR for amplification of del fragments AE and EG, cut

Conditions

Biorad MyCycler
Cycles-PCR	temperature [°C]	Time [s]
1	98	5
12	98	1
	66 ↓ 0.5	5
	72	2:30 min
18	98	1
	63	5
	72	2:30 min
1	72	10 min
1	4	inf

Results:

Amplification worked with 5% DMSO
Repeat Amplification with the same protocol to increase concentration when DNA is extracted from gel slices

03-07-2013

Amplification from FS_02 to FS_05; 11.2 kb

Amplification of DelA-F and DelF-G, 03-07-13; run at 130 V, 0.8 % gel (TAE)

Gel after removing the DelA-F-fragment, 03-07-13.

Reaction

µL 1st PCR	what	µL 2nd PCR
1	D. acidovorans DSM-39	1
2.5	FS_02 (1/10)	2.5
2.5	FS_05 (1/10)	2.5
25	Phusion Master Mix	25
-	DMSO	2.5
19	dd H₂O	19

Conditions

Biorad MyCycler
Cycles-PCR	temperature [°C]	Time [s]
1	98	10
12	98	1
	68 ↓ 0.5	5
	72	3 min
18	98	1
	66	5
	72	3 min
1	72	10 min
1	4	inf

Results:

Amplification of DelAE worked

bands were cut out and DNA purified using QIAquick Gel Extraction Kit

sample has to be purified before further use since contamination with propanol was present

04-07-2013

Amplification from FS_02 to FS_05; 11.2 kb

Gel electrophoresis of the PCR products of DelA-F (first three lane (counted from left)) and DelF-G (4th till 6th lane), 04-07-2013

Gel after removing DelA-F-fragment, 04-07-2013

Reaction

what	µL 2nd PCR
D. acidovorans DSM-39	1
FS_02 (1/10)	2.5
FS_05 (1/10)	2.5
Phusion Master Mix	25
DMSO	2.5
dd H₂O	16.5

Conditions

Biorad MyCycler
Cycles-PCR	temperature [°C]	Time [s]
1	98	10
12	98	1
	68 ↓ 0.5	5
	72	3 min
18	98	1
	66	5
	72	3 min
1	72	10 min
1	4	inf

Results:

Amplification of DelAE worked

bands were cut out and DNA purified using QIAquick Gel Extraction Kit resulting in a final concentratio of 10ng/µL

PCR will be repeated and gel slices purified with QIAX II Gel Extraction Kit, which is specifically designed to deliver higher yields when purifying fragments with sizes over 10 kbp

05-08-2013

Amplification from FS_02 to FS_07; 16.4 kb

Gel electrophoresis of DelA-G

Reaction

what	µl
D. acidovorans DSM-39	1
FS_02: (1/10)	1
FS_07: (1/10)	1
Phusion Master Mix	10
DMSO	1
dd H₂O	6

Conditions

Biorad MyCycler
Cycles-PCR	temperature [°C]	Time [s]
1	98	10
6	98	1
	66 ↓ 0.5	5
	72	5min 40s
24	98	1
	60	5
	72	5min 40s
1	72	17 min
1	4	inf

Results:

amplification of DelAG did not work
repeat PCR with higher annealing temperature to increase specifity

06-07-2013

Amplification from FS_04 to FS_07; 11.1 kb

PCR for amplification of del fragments E-G and A-E, AE+=PCR AE with DMSO, AE-=PCR without DMSO, M= Marker; wanted amplicon sizes are: AE=5.3kbp, EG=11.0

PCR for amplification of del fragments AE and EG, cut

Reaction

what	µl
D. acidovorans DSM-39	1
FS_04: (1/10)	1
FS_07: (1/10)	1
Phusion Master Mix	10
DMSO	1/-
dd H₂O	6/7

Conditions

Biorad MyCycler
Cycles-PCR	temperature [°C]	Time [s]
1	98	10
12	98	1
	68 ↓ 0.5	5
	72	3 min
18	98	1
	66	5
	72	3 min
1	72	10 min
1	4	inf

Results:

Amplification of DelEG worked but another unexpected product was amplified as well

band was cut out avoiding contamination with other product and DNA purified using QIAquick Gel Extraction Kit

03-07-2013

Amplification from FS_06 to FS_09; 8.5 kb

Amplification of DelA-F and DelF-G run at 130 V, 0.8 % gel (TAE)

Gel after removing the DelA-F-fragment.

Reaction

µl 1st PCR	what	µl 2nd PCR
1	D. acidovorans DSM-39	1
2.5	FS_06 (1/10)	2.5
2.5	FS_09 (1/10)	2.5
25	Phusion Master Mix	25
-	DMSO	2.5
19	dd H₂O	19

Conditions

Biorad MyCycler
Cycles-PCR	temperature [°C]	Time [s]
1	98	10
30	98	1
30	72	2:30 min
1	72	10 min
1	4	inf

Results:

Amplification of DelFG did not work
PCR will be repeated with a lower annealing temperature of 68°C (touchdown)

Amplification from FS_06 to FS_09; 8.5 kb

Gel electrophoresis of the PCR products of DelA-F (first three lane (counted from left)) and DelF-G (4th till 6th lane)

Gel after removing DelA-F-fragment

Reaction

what	µl 2nd PCR
D. acidovorans DSM-39	1
FS_06 (1/10)	2.5
FS_09 (1/10)	2.5
Phusion Master Mix	25
DMSO	2.5
dd H₂O	16.5

Conditions

Biorad MyCycler
Cycles-PCR	temperature [°C]	Time [s]
1	98	10
12	98	1
	68 ↓ 0.5	5
	72	3 min
18	98	1
	66	5
	72	3 min
1	72	10 min
1	4	inf

Results:

Amplification of DelFG did not work, many different side product occured, PCR will be repeated with other primers

Amplification from FS_06 to FS_11; 11.6 kb

Reaction

what	µl
D. acidovorans DSM-39	1
FS_06: (1/10)	1
FS_11: (1/10)	1
Phusion Master Mix	10
DMSO	1
dd H₂O	6

Conditions

Biorad MyCycler
Cycles-PCR	temperature [°C]	Time [s]
1	98	10
12	98	1
	68 ↓ 0.5	5
	72	3 min
18	98	1
	66	5
	72	3 min
1	72	10 min
1	4	inf

05-07-2013

Amplification from FS_06 to FS_07; 5.2 kb

Reaction

Gel electrophoresis of DelF-G, Backbone, DelF-G (5.1 kb), DelG (6.4 kb).

after Gel excision

what	µl
D. acidovorans DSM-39	1
FS_06: (1/10)	1
FS_07: (1/10)	1
Phusion Master Mix	10
DMSO	1
dd H₂O	6

Conditions

Biorad MyCycler
Cycles-PCR	temperature [°C]	Time [s]
1	98	5
14	98	1
	62 ↓ 0.5	5
	72	3:20 min
16	98	1
	60	5
	72	3:20 min
1	72	12 min
1	4	inf

05-07-2013

Amplification from FS_08 to FS_11; 6.5 kb

Gel electrophoresis of DelF-G, Backbone, DelF-G (5.1 kb), DelG (6.4 kb).

after Gel excision

Reaction

what	µl
D. acidovorans DSM-39	1
FS_08: (1/10)	1
FS_11: (1/10)	1
Phusion Master Mix	10
dd H₂O	6
DMSO	1

Conditions

Biorad MyCycler
Cycles-PCR	temperature [°C]	Time [s]
1	98	5
14	98	1
	62 ↓ 0.5	5
	72	3:20 min
16	98	1
	60	5
	72	3:20 min
1	72	12 min
1	4	inf

Results:

A weak band was visible, but it was on the wrong height.
Accidently the band was cut anyway.
Either the primers did not bind or the DNA still had to many secondary structures --> the consequence is to change the annealing temperature.

07-07-2013

Amplification from FS_08 to FS_11; 6.5 kb

PCR for amplification of del fragments DelAE (08.07), pSB4K5 (08.07) and DelG (07.07); lane1=Marker, lanes2-4=DelAE (5.3kbp), lanes5-10=pSB4K5 (4.2kbp), lanes11-13:DelG (6.4kbp); run at 100 V, 0.8 % gel (TAE)

PCR for amplification of del fragments DelAE (08.07), pSB4K5 (08.07) and DelG (07.07), cut

Reaction

what	µl
D. acidovorans DSM-39	1
FS_08: (1/10)	2.5
FS_11: (1/10)	2.5
Phusion Master Mix	25
dd H₂O	19
DMSO	2.5

Conditions

Biorad MyCycler
Cycles-PCR	temperature [°C]	Time [s]
1	98	5
12	98	1
	66 ↓ 0.5	5
	72	2:30 min
18	98	1
	63	5
	72	2:30 min
1	72	10 min
1	4	inf

Results:

There was no band visible on the gel.
Either the primers did not bind or the DNA still had to many secondary structures --> the consequence is to change the annealing temperature.

Overview
DelA-E

06-07-2013

Amplification from FS_02 to FS_03; 5.3 kb

PCR for amplification of del fragments E-G and A-E, AE+=PCR AE with DMSO, AE-=PCR without DMSO, M= Marker; wanted amplicon sizes are: AE=5.3kb, EG=11.0

Reaction

what	µL
D. acidovorans DSM-39	1
FS_02: (1/10)	1
FS_03: (1/10)	1
Phusion Master Mix	10
DMSO	1/-
dd H₂O	6/7

PCR for amplification of del fragments AE and EG, cut

Conditions

Biorad MyCycler
Cycles-PCR	temperature [°C]	Time [s]
1	98	5
12	98	1
	66 ↓ 0.5	5
	72	2:30 min
18	98	1
	63	5
	72	2:30 min
1	72	10 min
1	4	inf

Results:

Amplification worked with 5% DMSO
Repeat Amplification with the same protocol to increase concentration when DNA is extracted from gel slices

Overview
DelA-E

06-07-2013

Amplification from FS_02 to FS_03; 5.3 kb

PCR for amplification of del fragments E-G and A-E, AE+=PCR AE with DMSO, AE-=PCR without DMSO, M= Marker; wanted amplicon sizes are: AE=5.3kb, EG=11.0

Reaction

what	µL
D. acidovorans DSM-39	1
FS_02: (1/10)	1
FS_03: (1/10)	1
Phusion Master Mix	10
DMSO	1/-
dd H₂O	6/7

PCR for amplification of del fragments AE and EG, cut

Conditions

Biorad MyCycler
Cycles-PCR	temperature [°C]	Time [s]
1	98	5
12	98	1
	66 ↓ 0.5	5
	72	2:30 min
18	98	1
	63	5
	72	2:30 min
1	72	10 min
1	4	inf

Results:

Amplification worked with 5% DMSO
Repeat Amplification with the same protocol to increase concentration when DNA is extracted from gel slices

03-07-2013

Amplification from FS_02 to FS_05; 11.2 kb

Amplification of DelA-F and DelF-G, 03-07-13; run at 130 V, 0.8 % gel (TAE)

Gel after removing the DelA-F-fragment, 03-07-13.

Reaction

µL 1st PCR	what	µL 2nd PCR
1	D. acidovorans DSM-39	1
2.5	FS_02 (1/10)	2.5
2.5	FS_05 (1/10)	2.5
25	Phusion Master Mix	25
-	DMSO	2.5
19	dd H₂O	19

Conditions

Biorad MyCycler
Cycles-PCR	temperature [°C]	Time [s]
1	98	10
12	98	1
	68 ↓ 0.5	5
	72	3 min
18	98	1
	66	5
	72	3 min
1	72	10 min
1	4	inf

Results:

Amplification of DelAE worked

bands were cut out and DNA purified using QIAquick Gel Extraction Kit

sample has to be purified before further use since contamination with propanol was present

04-07-2013

Amplification from FS_02 to FS_05; 11.2 kb

Gel electrophoresis of the PCR products of DelA-F (first three lane (counted from left)) and DelF-G (4th till 6th lane), 04-07-2013

Gel after removing DelA-F-fragment, 04-07-2013

Reaction

what	µL 2nd PCR
D. acidovorans DSM-39	1
FS_02 (1/10)	2.5
FS_05 (1/10)	2.5
Phusion Master Mix	25
DMSO	2.5
dd H₂O	16.5

Conditions

Biorad MyCycler
Cycles-PCR	temperature [°C]	Time [s]
1	98	10
12	98	1
	68 ↓ 0.5	5
	72	3 min
18	98	1
	66	5
	72	3 min
1	72	10 min
1	4	inf

Results:

Amplification of DelAE worked

bands were cut out and DNA purified using QIAquick Gel Extraction Kit resulting in a final concentratio of 10ng/µL

PCR will be repeated and gel slices purified with QIAX II Gel Extraction Kit, which is specifically designed to deliver higher yields when purifying fragments with sizes over 10 kbp

Overview
DelA-E

06-07-2013

Amplification from FS_02 to FS_03; 5.3 kb

PCR for amplification of del fragments E-G and A-E, AE+=PCR AE with DMSO, AE-=PCR without DMSO, M= Marker; wanted amplicon sizes are: AE=5.3kb, EG=11.0

Reaction

what	µL
D. acidovorans DSM-39	1
FS_02: (1/10)	1
FS_03: (1/10)	1
Phusion Master Mix	10
DMSO	1/-
dd H₂O	6/7

PCR for amplification of del fragments AE and EG, cut

Conditions

Biorad MyCycler
Cycles-PCR	temperature [°C]	Time [s]
1	98	5
12	98	1
	66 ↓ 0.5	5
	72	2:30 min
18	98	1
	63	5
	72	2:30 min
1	72	10 min
1	4	inf

Results:

Amplification worked with 5% DMSO
Repeat Amplification with the same protocol to increase concentration when DNA is extracted from gel slices

Overview
DelA-E

06-07-2013

Amplification from FS_02 to FS_03; 5.3 kb

PCR for amplification of del fragments E-G and A-E, AE+=PCR AE with DMSO, AE-=PCR without DMSO, M= Marker; wanted amplicon sizes are: AE=5.3kb, EG=11.0

Reaction

what	µL
D. acidovorans DSM-39	1
FS_02: (1/10)	1
FS_03: (1/10)	1
Phusion Master Mix	10
DMSO	1/-
dd H₂O	6/7

PCR for amplification of del fragments AE and EG, cut

Conditions

Biorad MyCycler
Cycles-PCR	temperature [°C]	Time [s]
1	98	5
12	98	1
	66 ↓ 0.5	5
	72	2:30 min
18	98	1
	63	5
	72	2:30 min
1	72	10 min
1	4	inf

Results:

Amplification worked with 5% DMSO
Repeat Amplification with the same protocol to increase concentration when DNA is extracted from gel slices

Overview
DelA-E

06-07-2013

Amplification from FS_02 to FS_03; 5.3 kb

PCR for amplification of del fragments E-G and A-E, AE+=PCR AE with DMSO, AE-=PCR without DMSO, M= Marker; wanted amplicon sizes are: AE=5.3kb, EG=11.0

Reaction

what	µL
D. acidovorans DSM-39	1
FS_02: (1/10)	1
FS_03: (1/10)	1
Phusion Master Mix	10
DMSO	1/-
dd H₂O	6/7

PCR for amplification of del fragments AE and EG, cut

Conditions

Biorad MyCycler
Cycles-PCR	temperature [°C]	Time [s]
1	98	5
12	98	1
	66 ↓ 0.5	5
	72	2:30 min
18	98	1
	63	5
	72	2:30 min
1	72	10 min
1	4	inf

Results:

Amplification worked with 5% DMSO
Repeat Amplification with the same protocol to increase concentration when DNA is extracted from gel slices

Overview
DelA-E

06-07-2013

Amplification from FS_02 to FS_03; 5.3 kb

PCR for amplification of del fragments E-G and A-E, AE+=PCR AE with DMSO, AE-=PCR without DMSO, M= Marker; wanted amplicon sizes are: AE=5.3kb, EG=11.0

Reaction

what	µL
D. acidovorans DSM-39	1
FS_02: (1/10)	1
FS_03: (1/10)	1
Phusion Master Mix	10
DMSO	1/-
dd H₂O	6/7

PCR for amplification of del fragments AE and EG, cut

Conditions

Biorad MyCycler
Cycles-PCR	temperature [°C]	Time [s]
1	98	5
12	98	1
	66 ↓ 0.5	5
	72	2:30 min
18	98	1
	63	5
	72	2:30 min
1	72	10 min
1	4	inf

Results:

Amplification worked with 5% DMSO
Repeat Amplification with the same protocol to increase concentration when DNA is extracted from gel slices

Overview
DelA-E

06-07-2013

Amplification from FS_02 to FS_03; 5.3 kb

PCR for amplification of del fragments E-G and A-E, AE+=PCR AE with DMSO, AE-=PCR without DMSO, M= Marker; wanted amplicon sizes are: AE=5.3kb, EG=11.0

Reaction

what	µL
D. acidovorans DSM-39	1
FS_02: (1/10)	1
FS_03: (1/10)	1
Phusion Master Mix	10
DMSO	1/-
dd H₂O	6/7

PCR for amplification of del fragments AE and EG, cut

Conditions

Biorad MyCycler
Cycles-PCR	temperature [°C]	Time [s]
1	98	5
12	98	1
	66 ↓ 0.5	5
	72	2:30 min
18	98	1
	63	5
	72	2:30 min
1	72	10 min
1	4	inf

Results:

Amplification worked with 5% DMSO
Repeat Amplification with the same protocol to increase concentration when DNA is extracted from gel slices

Overview
DelA-E

06-07-2013

Amplification from FS_02 to FS_03; 5.3 kb

PCR for amplification of del fragments E-G and A-E, AE+=PCR AE with DMSO, AE-=PCR without DMSO, M= Marker; wanted amplicon sizes are: AE=5.3kb, EG=11.0

Reaction

what	µL
D. acidovorans DSM-39	1
FS_02: (1/10)	1
FS_03: (1/10)	1
Phusion Master Mix	10
DMSO	1/-
dd H₂O	6/7

PCR for amplification of del fragments AE and EG, cut

Conditions

Biorad MyCycler
Cycles-PCR	temperature [°C]	Time [s]
1	98	5
12	98	1
	66 ↓ 0.5	5
	72	2:30 min
18	98	1
	63	5
	72	2:30 min
1	72	10 min
1	4	inf

Results:

Amplification worked with 5% DMSO
Repeat Amplification with the same protocol to increase concentration when DNA is extracted from gel slices

Overview
DelA-E

06-07-2013

Amplification from FS_02 to FS_03; 5.3 kb

PCR for amplification of del fragments E-G and A-E, AE+=PCR AE with DMSO, AE-=PCR without DMSO, M= Marker; wanted amplicon sizes are: AE=5.3kb, EG=11.0

Reaction

what	µL
D. acidovorans DSM-39	1
FS_02: (1/10)	1
FS_03: (1/10)	1
Phusion Master Mix	10
DMSO	1/-
dd H₂O	6/7

PCR for amplification of del fragments AE and EG, cut

Conditions

Biorad MyCycler
Cycles-PCR	temperature [°C]	Time [s]
1	98	5
12	98	1
	66 ↓ 0.5	5
	72	2:30 min
18	98	1
	63	5
	72	2:30 min
1	72	10 min
1	4	inf

Results:

Amplification worked with 5% DMSO
Repeat Amplification with the same protocol to increase concentration when DNA is extracted from gel slices

Overview
DelA-E

08-07-2013

Amplification from FS_02 to FS_03; 5.3 kb

PCR for amplification of del fragments DelAE (08.07), pSB4K5 (08.07) and DelG (07.07); lane1=Marker, lanes2-4=DelAE (5.3kb), lanes5-10=pSB4K5 (4.2kb), lanes11-13:DelG (6.4kb); run at 100 V, 0.8 % gel (TAE)

Reaction

what	µL
D. acidovorans DSM-39	1
FS_02: (1/10)	2.5
FS_03: (1/10)	2.5
Phusion Master Mix	25
DMSO	2.5
dd H₂O	19

PCR for amplification of del fragments DelAE (08.07), pSB4K5 (08.07) and DelG (07.07), cut

Conditions

Biorad MyCycler
Cycles-PCR	temperature [°C]	Time [s]
1	98	5
12	98	1
	66 ↓ 0.5	5
	72	2:30 min
18	98	1
	63	5
	72	2:30 min
1	72	10 min
1	4	inf

Results:

Amplification of DelAE worked
bands were cut out and DNA purified using QIAquick Gel Extraction Kit

Overview
DelA-E

19-07-2013

Amplificaction from FS_02 to FS_03; 5.3 kb

+=with DMSO, -=without DMSO; expected amplicon sizes: LP=6.4kb, 8-9=3.1kb, 21-09 and 20-09=8.1kb, 22-13s=2.6kb, AE=5.3kb; run at 100 V, 0.8 % gel (TAE)

Amplifications of 18.07 and 19.07, cut

Reaction

2x ~50 µL

what	µL
D. acidovorans DSM-39	1
FS_02: (1/10)	2.5
FS_03: (1/10)	2.5
Phusion flash Master Mix	25
DMSO	2.5
dd H₂O	19

Conditions

Biometra TProfessional Basic
Cycles	temperature [°C]	Time [s]
1	98	5
12	98	1
	66 ↓ 0.5	5
	72	2:30
18	98	1
	63	5
	72	2:30
1	72	10min
1	12	inf

Results:

Amplification of DelAE did not work since a different cycler was used

20-07-2013

Amplification from FS_02 to FS_03; 5.3 kb

Amplification of AE, OP, FG (20.07); expected amplicon sizes: 21-09(FG)=8.1kb, 22-13s(OP)=2.6kb, AE=5.3kb; run at 100 V, 0.8 % gel (TAE)

Reaction

what	µL
D. acidovorans DSM-39	1
FS_02: (1/10)	2.5
FS_03: (1/10)	2.5
Phusion flash Master Mix	25
DMSO	2,5
dd H₂O	19

Conditions

Biorad MyCycler
Cycles	temperature [°C]	Time [s]
1	98	5
12	98	1
	66 ↓ 0.5	5
	72	2:30
18	98	1
	63	5
	72	2:30
1	72	10min
1	12	inf

Results:

Amplification of DelAE worked but a smear occured, therefore bands were cut out carefully and only used for a test restriction digest

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Overview
DelA-E

26-07-2013

Restriction digest of fragment FS_02 to FS_03; 5.3 kb; 08-07-2013 with EcoRI-HF

Restriction digest of DelAE (FS_02 to FS_03 ;08.07), DelAF (FS_02 to FS_05; 04.07) and DelOP (FS_22 to FS_13short ;21.07) with EcoRI-HF and digest of DelG (FS_08 to FS_23 cut1 ;23.07) with BglII; run at 100 V, 0.8 % gel (TAE)

Incubation at 37°C for 1 h 45 min

what	µL
FS_02 to FS_03 (08-07-2013)	15
EcorRI-HF	0.5
Buffer CutSmart	2
dd H₂O	2
Expected fragment lengths [bp]	3054, 2260

Results:

restriction digest of DelAE did not work, since incubation time might have been to short

27-07-2013

Amplificaction from FS_02 to FS_03; 5.3 kb

Amplification of DelAE (FS02 to FS03; 27.07,1), Amplification of DelAF (FS_02 to FS05; 27.07); run at 100 V, 0.8 % gel (TAE)

Amplification of DelAE (FS02 to FS03; 27.07,1), Amplification of DelAF (FS_02 to FS05; 27.07), cut; run at 100 V, 0.8 % gel (TAE)

Reaction

what	µL
D. acidovorans DSM-39	1
FS_02: (1/10)	2.5
FS_03: (1/10)	2.5
Phusion flash Master Mix	25
DMSO	2.5
dd H₂O	19

Conditions

Biorad MyCycler
Cycles	temperature [°C]	Time [s]
1	98	10
12	98	1
	66 ↓ 0.5	5
	72	2:30
18	98	1
	63	5
	72	2:30
1	72	12 min
1	12	inf

Results:

Amplification of DelAE didnt work out, only smear occured
repeat PCR with better cycler

Amplificaction from FS_02 to FS_03; 5.3 kb

2nd Amplification of DelAE (FS02 to FS03; 27.07,1), first two lanes with 5 µL primer, second two lanes with 2.5 µL primer; run at 100 V, 0.8 % gel (TAE)

gel after excision

Reaction

what	µL
D. acidovorans DSM-39	1.5/1
FS_02: (1/10)	2.5/5
FS_03: (1/10)	2.5/5
Phusion flash Master Mix	25
DMSO	2.5
dd H₂O	19/14

Conditions

Biorad MyCycler*
Cycles	temperature [°C]	Time [s]
1	98	10
12	98	1
	66 ↓ 0.5	5
	72	2:30
18	98	1
	63	5
	72	2:30
1	72	12 min
1	12	inf

Results:

Amplification of DelAE worked with both 200 and 400 nM of Primers, nevertheless amplification was more specific with the higher primer concentration
bands were cut out and DNA purified using QIAquick Gel Extraction Kit

Amplification I from FS_02 to FS_24; 7.1 kb

Amplifikation of DelFG (FS02-FS24), ; run at 100 V, 0.8 % gel (TAE)

4 reactions, 2 with 200nM Primers and 2 with 400nM Primers (both concentrations for each condition)

Reaction

what	µl
D. acidovorans DSM-39	1
FS_02: (1/10)	4/2
FS_24: (1/10)	4/2
Phusion flash Master Mix	10
DMSO	1
dd H₂O	0/4

Conditions I

Biorad T100
Cycles	temperature [°C]	Time [s]
1	98	10
30	98	1
	65	5
	72	3:50
1	72	12 min
1	10	inf

Conditions II

Biometra TProfessional Basic
Cycles-PCR	temperature [°C]	Time [s]
1	98	10
12	98	1
	68 ↓ 0.5	5
	72	3:50 min
18	98	1
	66	5
	72	3:50 min
1	72	10 min
1	4	inf

Results:

Amplification of DelAE worked with 200 nM primer concentration at an annealing temperature of 68°C and 400 nM at an annealing temperature of 65°C, the product obtained at 65°C was more specific
bands were cut out and DNA purified using QIAquick Gel Extraction Kit

Amplification II from FS_02 to FS_24; 7.1 kb

2 x Amplification of FS02 to FS_24 27.07.13 68°C Touchdown 200nM/400nM; 4x Amplification of FS-02 to FS23 27.0713 1 & 2 68°C Touchdown 200nM/400nM; 3. & 4. 65°C const. 200/400 nM, run at 100V

2 reactions, 68°C Touchdown with 200nM Primers and 65°C constant with 400nM Primers

Reaction

what	µl
D. acidovorans DSM-39	1
FS_02: (1/10)	2/4
FS_24: (1/10)	2/4
Phusion flash Master Mix	10
DMSO	1
dd H₂O	0/4

Conditions I

Biorad T100
Cycles	temperature [°C]	Time [s]
1	98	10
30	98	1
	65	5
	72	5:40
1	72	12 min
1	10	inf

Conditions II

Biometra TProfessional Basic
Cycles-PCR	temperature [°C]	Time [s]
1	98	10
12	98	1
	68 ↓ 0.5	5
	72	5:40 min
18	98	1
	66	5
	72	5:40 min
1	72	10 min
1	4	inf

Results:

Amplification did not work, neither with 200nM and 68°C touchdown, nor with 400nM and 65°C constant.
Repeat amplfication with different conditions as primers did not bind very effectively

Amplification III from FS_02 to FS_24; 7.1 kb

1kbruler / 4xFS02-FS23 60const @T100 200nM; T100 400nM; old 200nM; old 400nM ; red 200nM; red 400nM / FS02-FS24 66td janusA 200nM / FS02-FS24 T100 60const 200nM; 400nM 1kb FS04-FS24.png ; run at 100 V, 0.8 % gel (TAE)

2 reactions, 66°C Touchdown with 200nM Primers and 60°C constant with 400nM Primers

Reaction

what	µl
D. acidovorans DSM-39	1
FS_02: (1/10)	2/4
FS_24: (1/10)	2/4
Phusion flash Master Mix	10
DMSO	1
dd H₂O	0/4

Conditions I

Biorad T100
Cycles	temperature [°C]	Time [s]
1	98	10
30	98	1
	60	5
	72	5:40
1	72	12 min
1	10	inf

Conditions II

Biorad C1000 Touch Block A
Cycles-PCR	temperature [°C]	Time [s]
1	98	10
12	98	1
	66 ↓ 0.5	5
	72	5:40 min
18	98	1
	64	5
	72	5:40 min
1	72	10 min
1	4	inf

Results:

Amplification from DelAE (7.1 kbp) failed again
stick to the old strategy and use previously obtained fragments with different other fragments for gibson assembly.

29-07-2013

Restriction digest of FS_02 to FS_03; 5.3 kb;(27-07-2013; II) with BglII

Test restriction digest (29.07); run at 100 V, 0.8 % gel (TAE)

Incubation at 37°C for about 3 h

what	µL
FS_02 to FS_03 (27-07-2013; II)	15
BglII	1
Buffer 3.1	2
dd H₂O	2

Expected fragment lengths: 2,146 kb; 1,862 kb; 1,306 kb

Results:

Restriction digest shows the expected product sizes
indicator for correct amplicon but to be sure, PCR product will be prepared for single read sequencing by GATC

Overview
DelA-E

29-07-2013

Restriction digest of fragment FS_02 to FS_03 (5.3 kb; 27-07-2013; II) with BglII

Test restriction digest (29.07); run at 100 V, 0.8 % gel (TAE)

Incubation at 37°C for about 3 h

what	µL
FS_02 to FS_03 (27-07-2013; II)	15
BglII	1
Buffer 3.1	2
dd H₂O	2
Expected fragment lengths [bp]	2146, 1862, 1306

Results:

Restriction digest shows the expected product sizes
indicator for correct amplicon but to be sure, PCR product will be prepared for single read sequencing by GATC

Overview
DelA-E

07-08-2013

Sequencing Results

We send the following samples (amplified fragments of D. acidovorans DSM-39 and backbone pSB4K5 from the partsregistry) to GATC for sequencing:

AE (FS_02-FS_03) with Primer FS_02
AE (FS_02-FS_03) with Primer FS_03

Alignment of DelAE(FS_02 to FS_03) with Primer FS_02

Alignment of DelAE(FS_02 to FS_03) with Primer FS_03

08-08-2013

Amplification from FS_02 to FS_03; 5.3kb

Amplifcation of DelAE and DelEF and DelOP; run at 100 V, 0.8 % gel (TAE)(07.08)

Reaction

what	µL
D. acidovorans SPH-1	1
FS_02: (1/10)	4
FS_03: (1/10)	4
Phusion flash Master Mix	10

Conditions

Biometra TProfessional Basic
Cycles	temperature [°C]	Time [s]
1	98	10
30	98	1
	58	5
	72	1:30
1	72	7min
1	12	inf

Results:

Amplification of DelAE was repeated successfully with the new strain SPH-1
bands were cut out and DNA purified using QIAquick Gel Extraction Kit
if fragment is used in Gibson assembly the amplification has to be repeated to increase the amount of product

Overview
DelA-F

12-08-2013

Amplification from FS_02 to FS_05; 11.2 kb

Amplification of DelAF using gradient PCR, Amplification of DelOP 72°C 2-step; run at 135 V, 0.8 % gel (TAE)(11.08)

Amplification of DelAF using gradient PCR, Amplification of DelOP 72°C 2-step after cutting; run at 135 V, 0.8 % gel (TAE)(11.08)

Reaction

Reagent	DelAF
Template	D.acidovorans SPH-1 colony
Primer fw	4.5 µL FS_02
Primer rev	4.5 µL FS_05
DMSO	1 µL
Phusion Ready Mix	10 µL

Conditions

Biometra TProfessional Basic
Cycles	temperature [°C]	Time [s]
1	98	10
12	98	1
	67.5 - 65.0 (ΔT = 0.5) ↓ 0.5	5
	72	3:20
18	98	1
	65.5 - 63.0 (ΔT = 0.5)	5
	72	3:20
1	72	10min
1	10	inf

Results:

Amplification of DelAF worked well, gradient displays an optimal annealing temperature of 65.5°C, which will be used for further amplifications
bands were cut out and DNA purified using QIAquick Gel Extraction Kit

13-08-2013

Restriction digest of fragment FS_02 to FS_05; 11.2 kb; 12-08-2013 with EcoRI-HF

Incubation at 37°C for 2 hours

what	µL
FS_02 to FS_05 (12-08-2013)	20
EcoRI-HF	1
CutSmart Buffer	2.5
dd H₂O	1.5

Expected fragment sizes: 2.26kbp; 4.62kbp; 4.32kbp

Results:

Weak bands of about 4.6kbp visible, as well as on of about 2.6kbp
digest will be repeated using higher concentrations of DNA to clearify results of restriction digest

Amplification from FS_02 to FS_05; 11.2 kb

Amplification of DelAF (FS_02-FS_05); run at 100 V, 0.8 % gel (TAE)(13.08)

Amplification of DelAF (FS_02-FS_05), cut; run at 100 V, 0.8 % gel (TAE)(13.08)

Reaction

1 sample contains 2µL of DMSO

Reagent	DelAF
Template	D.acidovorans SPH-1 colony
Primer fw	4.5 µL FS_02
Primer rev	4.5 µL FS_05
DMSO	1 µL
Phusion Ready Mix	10 µL

Conditions

Biorad T100
Cycles	temperature [°C]	Time [s]
1	98	10
12	98	1
	65.5 ↓ 0.5	5
	72	3:20
18	98	1
	63.5	5
	72	3:20
1	72	10min
1	10	inf

Results:

Amplification of DelAF worked though a slight smear occured, therefore PCR will be repeated on the more precise Biometra TProfessional Basic cylcer again
bands were cut out and DNA purified using QIAquick Gel Extraction Kit

14-08-2013

Concentration measurement (FS_02 to FS_05; 11.2 kb)

Fragment	Primer	Date PCR	Concentration
DelAF	FS02-FS05	11-08-2013	~10 ng/µL
DelAF	FS02-FS05	12-08-2013	0 ng/µL
DelAF	FS02-FS05	12-08-2013	0 ng/µL

Results:

concentrations are not sufficient for gibson assembly
PCR will be repeated and gel slices of different reactions will be pooled for one gel extraction using QIAquick Gel Extraction Kit to obtain the concentrations needed for gibson assembly

Restriction digest of fragment FS_02 to FS_05; 11.2 kb; 11-08-2013 with EcoRI-HF

Test restriction digest of fragment FS_02-FS_05 (12-08) with EcoRI; run at 100 V, 0.8 % gel (TAE)(13.08)

Incubation at 37°C for 2 hours 45min

what	µL
FS_02 to FS_05 (11-08-2013)	18
EcoRI-HF	1
CutSmart Buffer	2.5
dd H₂O	3.5

Expected fragment sizes: 2.26kbp; 4.62kbp; 4.32kbp

Results:

One band of about 5kbp and one of 2.5kbp
no clear result, digest will be repeated with another enzyme (ClaI), as the enzyme used was beyond expiration date

Restriction digest of fragment FS_02 to FS_05; 11.2 kb; 10-08-2013 with ClaI

Test restriction digest of fragment FS_02-FS_05 (12-08) with ClaI; run at 100 V, 0.8 % gel (TAE)(14.08)

Incubation at 37°C for 2 hours

what	µL
FS_02 to FS_05 (10-08-2013)	20
ClaI	1
CutSmart Buffer	2.5
dd H₂O	1.5

Expected fragment sizes: 6.9kbp, 4.3kbp

Results:

restriction digest displays the expected fragments, therefore amplification of the desired fragment can be assumed
PCR product will be prepared for sequencing by GATC to proof amplification of the desired DNA sequence before Gibson Assembly

17-08-2013

Amplification from FS_02 to FS_05; 11.2 kb

Amplification of DelAF (17-08); run at 100 V, 0.8 % gel (TAE)

Amplification of DelAF, cut (17-08); run at 100 V, 0.8 % gel (TAE)

Reaction of DelAF

6x20 µL

Reagent	DelAF
Template	D.acidovorans SPH-1 colony
Primer fw	2.5 µL FS_02
Primer rev	2.5 µL FS_05
Phusion Ready Mix	10 µL
DMSO	1 µL
dd H₂O	4 µL

Conditions

Biometra TProfessional Basic
Cycles	temperature [°C]	Time [s]
1	98	10
12	98	1
	65.5 ↓ 0.5	5
	72	3:20
18	98	1
	63.5	5
	72	3:20
1	72	10min
1	10	inf

Results:

Amplification of DelAF worked
bands were cut out, pooled and DNA purified using QIAquick Gel Extraction Kit to obtain concentrations needed for gibson assembly

18-08-2013

Amplification from FS_02 to FS_05; 11.2 kb

Amplification of DelAF (18-08); run at 100 V, 0.8 % gel (TAE)

Amplification of DelAF, cut (18-08); run at 100 V, 0.8 % gel (TAE)

Reaction of DelAF

6x20 µL

Reagent	DelAF
Template	D.acidovorans SPH-1 colony
Primer fw	2.5 µL FS_02
Primer rev	2.5 µL FS_05
DMSO	1 µL
Phusion Ready Mix	10 µL
dd H₂O	4 µL

Conditions

Biometra TProfessional Basic
Cycles	temperature [°C]	Time [s]
1	98	10
12	98	1
	65.5 ↓ 0.5	5
	72	3:20
18	98	1
	63.5	5
	72	3:20
1	72	10min
1	10	inf

Results:

Amplification of DelAF worked
bands were cut out and DNA purified using QIAquick Gel Extraction Kit

Overview
pSB4K5

19-08-2013

Concentration measurement

The concentration of the gel purified, and DpnI digested fragments was measured using a NanoDrop Instrument.

Fragment	Primer	Date PCR	Concentration
pSB4K5 DpnI digested	FS_01-FS_16	17-08-2013	101.2 ng/µl
pSB4K5 DpnI digested	FS_01-FS_16	18-08-2013	159.2 ng/µl

Overview
pSB4K5

19-08-2013

Concentration measurement

The concentration of the gel purified, and DpnI digested fragments was measured using a NanoDrop Instrument.

Fragment	Primer	Date PCR	Concentration
pSB4K5 DpnI digested	FS_01-FS_16	17-08-2013	101.2 ng/µl
pSB4K5 DpnI digested	FS_01-FS_16	18-08-2013	159.2 ng/µl

Overview
pSB4K5

19-08-2013

Concentration measurement

The concentration of the gel purified, and DpnI digested fragments was measured using a NanoDrop Instrument.

Fragment	Primer	Date PCR	Concentration
pSB4K5 DpnI digested	FS_01-FS_16	17-08-2013	101.2 ng/µl
pSB4K5 DpnI digested	FS_01-FS_16	18-08-2013	159.2 ng/µl

Overview
pSB4K5

19-08-2013

Concentration measurement

The concentration of the gel purified, and DpnI digested fragments was measured using a NanoDrop Instrument.

Fragment	Primer	Date PCR	Concentration
pSB4K5 DpnI digested	FS_01-FS_16	17-08-2013	101.2 ng/µl
pSB4K5 DpnI digested	FS_01-FS_16	18-08-2013	159.2 ng/µl

Overview
pSB4K5

19-08-2013

Concentration measurement

The concentration of the gel purified, and DpnI digested fragments was measured using a NanoDrop Instrument.

Fragment	Primer	Date PCR	Concentration
pSB4K5 DpnI digested	FS_01-FS_16	17-08-2013	101.2 ng/µl
pSB4K5 DpnI digested	FS_01-FS_16	18-08-2013	159.2 ng/µl

Overview
pSB4K5

19-08-2013

Concentration measurement

The concentration of the gel purified, and DpnI digested fragments was measured using a NanoDrop Instrument.

Fragment	Primer	Date PCR	Concentration
pSB4K5 DpnI digested	FS_01-FS_16	17-08-2013	101.2 ng/µl
pSB4K5 DpnI digested	FS_01-FS_16	18-08-2013	159.2 ng/µl

Overview
pSB4K5

19-08-2013

Concentration measurement

The concentration of the gel purified, and DpnI digested fragments was measured using a NanoDrop Instrument.

Fragment	Primer	Date PCR	Concentration
pSB4K5 DpnI digested	FS_01-FS_16	17-08-2013	101.2 ng/µl
pSB4K5 DpnI digested	FS_01-FS_16	18-08-2013	159.2 ng/µl

@@ Line 358: / Line 358: @@
              <!--Start Weekly Labjournal-->
-            <!--Week 10-->
+          <!--Week 1-->
              <div class="labjournal-weekly">
                  <div>
                      <ul class="nav nav-tabs">
-                       <li class="active"><a href="#a10" data-toggle="tab">Overview</a></li>
+                       <li class="active"><a href="#a1" data-toggle="tab">Overview</a></li>
-                       <li><a href="#b10" data-toggle="tab">DelA-E</a></li>
+                       <li><a href="#b1" data-toggle="tab">DelA-E</a></li>
-                       <li><a href="#c10" data-toggle="tab">DelA-F</a></li>
+                       <li><a href="#c1" data-toggle="tab">DelA-F</a></li>
-                       <li><a href="#d10" data-toggle="tab">DelA-G</a></li>
+                       <li><a href="#d1" data-toggle="tab">DelA-G</a></li>
-                       <li><a href="#e10" data-toggle="tab">DelE-G</a></li>
+                       <li><a href="#e1" data-toggle="tab">DelE-G</a></li>
-                       <li><a href="#j10" data-toggle="tab">DelF-G</a></li>
+                       <li><a href="#j1" data-toggle="tab">DelF-G</a></li>
-                       <li><a href="#f10" data-toggle="tab">DelG</a></li>
+                       <li><a href="#f1" data-toggle="tab">DelG</a></li>
-                       <li><a href="#g10" data-toggle="tab">DelO-P</a></li>
+                       <li><a href="#g1" data-toggle="tab">DelO-P</a></li>
-                       <li><a href="#h10" data-toggle="tab">DelL</a></li>
+                       <li><a href="#h1" data-toggle="tab">DelL</a></li>
-                       <li><a href="#i10" data-toggle="tab">pSB4K5</a></li>
+                       <li><a href="#i1" data-toggle="tab">pSB4K5</a></li>
                      </ul>
                  </div>
@@ Line 379: / Line 379: @@
                          <div class="tab-content">
-                             <div class="tab-pane active" id="a10">
+                             <div class="tab-pane active" id="a1">
                                  <p style="font-size:12pt; text-align:justify;">
@@ Line 385: / Line 385: @@
                              </div>
-                             <div class="tab-pane" id="b10">
+                             <div class="tab-pane" id="b1">
                                  <p style="font-size:12pt; text-align:justify;">
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                                  </p>
                              </div>
-                             <div class="tab-pane" id="c10">
+                             <div class="tab-pane" id="c1">
                                  <p style="font-size:12pt; text-align:justify;">
                                  </html>{{:Team:Heidelberg/Templates/Del_week10_AF}}<html>
@@ Line 396: / Line 396: @@
                              </div>
-                             <div class="tab-pane" id="d10">
+                             <div class="tab-pane" id="d1">
                                  <p style="font-size:12pt; text-align:justify;">
@@ Line 403: / Line 403: @@
                              </div>
-                             <div class="tab-pane" id="e10">
+                             <div class="tab-pane" id="e1">
                                  <p style="font-size:12pt; text-align:justify;">
                                 </html>{{:Team:Heidelberg/Templates/Del_week10_EG}}<html>
@@ Line 409: / Line 409: @@
                              </div>
-                             <div class="tab-pane" id="j10">
+                             <div class="tab-pane" id="j1">
                                  <p style="font-size:12pt; text-align:justify;">
                                 </html>{{:Team:Heidelberg/Templates/Del_week10_FG}}<html>
@@ Line 415: / Line 415: @@
                              </div>
-                             <div class="tab-pane" id="f10">
+                             <div class="tab-pane" id="f1">
                                  <p style="font-size:12pt; text-align:justify;">
                                 </html>{{:Team:Heidelberg/Templates/Del_week10_G}}<html>
@@ Line 421: / Line 421: @@
                              </div>
-                             <div class="tab-pane" id="g10">
-                                <p style="font-size:12pt; text-align:justify;">
+                        </div>
-                               </html>{{:Team:Heidelberg/Templates/Del_week10_OP}}<html>
+                    </div>
-                                </p>
+                </div>
-                            </div>
+            </div>
-                             <div class="tab-pane" id="h10">
+         <!--Week 2-->
+            <div class="labjournal-weekly">
+                <div>
+                    <ul class="nav nav-tabs">
+                      <li class="active"><a href="#a2" data-toggle="tab">Overview</a></li>
+                      <li><a href="#b2" data-toggle="tab">DelA-E</a></li>
+                    </ul>
+                </div>
+                <div class="jumbotron">
+                    <div class="scrollContent nav navbar" data-spy="scroll" data-target="#navbarExample" data-offset="0">
+                        <div class="tab-content">
+                             <div class="tab-pane active" id="a2">
                                  <p style="font-size:12pt; text-align:justify;">
-                                </html>{{:Team:Heidelberg/Templates/Del_week10_L}}<html>
                                  </p>
                              </div>
-                             <div class="tab-pane" id="i10">
+                             <div class="tab-pane" id="b2">
                                  <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week10_pSB4K5}}<html>
+                                     </html>{{:Team:Heidelberg/Templates/Del_week10_AE}}<html>
                                  </p>
                              </div>
                          </div>
@@ Line 444: / Line 460: @@
              </div>
-            <!--Week 11-->
+         <!--Week 3-->
              <div class="labjournal-weekly">
                  <div>
                      <ul class="nav nav-tabs">
-                       <li class="active"><a href="#a11" data-toggle="tab">Overview</a></li>
+                       <li class="active"><a href="#a3" data-toggle="tab">Overview</a></li>
-                       <li><a href="#b11" data-toggle="tab">DelA-E</a></li>
+                       <li><a href="#b3" data-toggle="tab">DelA-E</a></li>
-                      <li><a href="#d11" data-toggle="tab">DelA-G</a></li>
-                      <li><a href="#e11" data-toggle="tab">DelE-G</a></li>
-                      <li><a href="#c11" data-toggle="tab">DelF-G</a></li>
-                      <li><a href="#f11" data-toggle="tab">DelG</a></li>
-                      <li><a href="#g11" data-toggle="tab">DelO-P</a></li>
-                      <li><a href="#j11" data-toggle="tab">DelL-P</a></li>
-                      <li><a href="#h11" data-toggle="tab">DelL</a></li>
-                      <li><a href="#i11" data-toggle="tab">pSB4K5</a></li>
                      </ul>
                  </div>
-                 <div class="jumbotron weekly">
+                 <div class="jumbotron">
                      <div class="scrollContent nav navbar" data-spy="scroll" data-target="#navbarExample" data-offset="0">
-                       <div class="tab-content">
-                            <div class="tab-pane active" id="a11">
-                                <p style="font-size:12pt; text-align:justify;">
-                                </p>
-                            </div>
-                             <div class="tab-pane" id="b11">
+                        <div class="tab-content">
+                             <div class="tab-pane active" id="3">
                                  <p style="font-size:12pt; text-align:justify;">
-                                     </html>{{:Team:Heidelberg/Templates/Del_week11_AE}}<html>
                                  </p>
                              </div>
-                             <div class="tab-pane" id="d11">
+                             <div class="tab-pane" id="b3">
                                  <p style="font-size:12pt; text-align:justify;">
+                                     </html>{{:Team:Heidelberg/Templates/Del_week10_AE}}<html>
-                                </html>{{:Team:Heidelberg/Templates/Del_week11_AG}}<html>
                                  </p>
                              </div>
+                             <div class="tab-pane" id="c3">
-                             <div class="tab-pane" id="e11">
                                  <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week11_EG}}<html>
+                                </html>{{:Team:Heidelberg/Templates/Del_week10_AF}}<html>
                                  </p>
                              </div>
-                             <div class="tab-pane" id="f11">
+                        </div>
+                    </div>
+                </div>
+            </div>
+        <!--Week 4-->
+            <div class="labjournal-weekly">
+                <div>
+                    <ul class="nav nav-tabs">
+                      <li class="active"><a href="#a4" data-toggle="tab">Overview</a></li>
+                      <li><a href="#b4" data-toggle="tab">DelA-E</a></li>
+                    </ul>
+                </div>
+                <div class="jumbotron">
+                    <div class="scrollContent nav navbar" data-spy="scroll" data-target="#navbarExample" data-offset="0">
+                        <div class="tab-content">
+                             <div class="tab-pane active" id="a4">
                                  <p style="font-size:12pt; text-align:justify;">
-                                </html>{{:Team:Heidelberg/Templates/Del_week11_G}}<html>
                                  </p>
                              </div>
-                             <div class="tab-pane" id="j11">
+                             <div class="tab-pane" id="b4">
                                  <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week11_LP}}<html>
+                                     </html>{{:Team:Heidelberg/Templates/Del_week10_AE}}<html>
                                  </p>
                              </div>
-                             <div class="tab-pane" id="g11">
+                        </div>
+                    </div>
+                </div>
+            </div>
+         <!--Week 5-->
+            <div class="labjournal-weekly">
+                <div>
+                    <ul class="nav nav-tabs">
+                      <li class="active"><a href="#a5" data-toggle="tab">Overview</a></li>
+                      <li><a href="#b5" data-toggle="tab">DelA-E</a></li>
+                    </ul>
+                </div>
+                <div class="jumbotron">
+                    <div class="scrollContent nav navbar" data-spy="scroll" data-target="#navbarExample" data-offset="0">
+                        <div class="tab-content">
+                             <div class="tab-pane active" id="a5">
                                  <p style="font-size:12pt; text-align:justify;">
-                                </html>{{:Team:Heidelberg/Templates/Del_week11_OP}}<html>
                                  </p>
                              </div>
-                             <div class="tab-pane" id="h11">
+                             <div class="tab-pane" id="b5">
                                  <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week11_L}}<html>
+                                     </html>{{:Team:Heidelberg/Templates/Del_week10_AE}}<html>
                                  </p>
                              </div>
-                             <div class="tab-pane" id="i11">
+                        </div>
+                    </div>
+                </div>
+            </div>
+         <!--Week 6-->
+            <div class="labjournal-weekly">
+                <div>
+                    <ul class="nav nav-tabs">
+                      <li class="active"><a href="#a6" data-toggle="tab">Overview</a></li>
+                      <li><a href="#b6" data-toggle="tab">DelA-E</a></li>
+                    </ul>
+                </div>
+                <div class="jumbotron">
+                    <div class="scrollContent nav navbar" data-spy="scroll" data-target="#navbarExample" data-offset="0">
+                        <div class="tab-content">
+                             <div class="tab-pane active" id="a6">
                                  <p style="font-size:12pt; text-align:justify;">
-                                </html>{{:Team:Heidelberg/Templates/Del_week11_pSB4K5}}<html>
                                  </p>
                              </div>
-                             <div class="tab-pane" id="c11">
+                             <div class="tab-pane" id="b6">
                                  <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week11_FG}}<html>
+                                     </html>{{:Team:Heidelberg/Templates/Del_week10_AE}}<html>
                                  </p>
                              </div>
-                       </div>
+                        </div>
-                   </div>
+                    </div>
-               </div>
+                </div>
              </div>
-                    <!--Week 12-->
+         <!--Week 7-->
              <div class="labjournal-weekly">
                  <div>
                      <ul class="nav nav-tabs">
-                       <li class="active"><a href="#a12" data-toggle="tab">Overview</a></li>
+                       <li class="active"><a href="#a7" data-toggle="tab">Overview</a></li>
-                       <li><a href="#b12" data-toggle="tab">DelA-E</a></li>
+                       <li><a href="#b7" data-toggle="tab">DelA-E</a></li>
-                      <li><a href="#e12" data-toggle="tab">DelE-G</a></li>
-                      <li><a href="#c12" data-toggle="tab">DelF-G</a></li>
-                      <li><a href="#f12" data-toggle="tab">DelG</a></li>
-                      <li><a href="#h12" data-toggle="tab">DelL-P</a></li>
-                      <li><a href="#g12" data-toggle="tab">DelO-P</a></li>
                      </ul>
                  </div>
-                    <div class="jumbotron weekly">
+                <div class="jumbotron">
                      <div class="scrollContent nav navbar" data-spy="scroll" data-target="#navbarExample" data-offset="0">
                          <div class="tab-content">
-                             <div class="tab-pane active" id="a12">
+                             <div class="tab-pane active" id="a7">
                                  <p style="font-size:12pt; text-align:justify;">
                                  </p>
                              </div>
-                             <div class="tab-pane" id="b12">
+                             <div class="tab-pane" id="b7">
                                  <p style="font-size:12pt; text-align:justify;">
-                                      </html>{{:Team:Heidelberg/Templates/Del_week12_AE}}<html>
+                                      </html>{{:Team:Heidelberg/Templates/Del_week10_AE}}<html>
                                  </p>
                              </div>
+                        </div>
+                    </div>
+                </div>
+            </div>
-                             <div class="tab-pane" id="e12">
+           <!--Week 8-->
+            <div class="labjournal-weekly">
+                <div>
+                    <ul class="nav nav-tabs">
+                      <li class="active"><a href="#a8" data-toggle="tab">Overview</a></li>
+                      <li><a href="#b8" data-toggle="tab">DelA-E</a></li>
+                    </ul>
+                </div>
+                <div class="jumbotron">
+                    <div class="scrollContent nav navbar" data-spy="scroll" data-target="#navbarExample" data-offset="0">
+                        <div class="tab-content">
+                             <div class="tab-pane active" id="a8">
                                  <p style="font-size:12pt; text-align:justify;">
-                                </html>{{:Team:Heidelberg/Templates/Del_week12_EG}}<html>
                                  </p>
                              </div>
-                             <div class="tab-pane" id="f12">
+                             <div class="tab-pane" id="b8">
                                  <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week12_G}}<html>
+                                     </html>{{:Team:Heidelberg/Templates/Del_week10_AE}}<html>
-                                </p>
-                            </div>
-                            <div class="tab-pane" id="h12">
-                                <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week12_LP}}<html>
                                  </p>
                              </div>
-                             <div class="tab-pane" id="g12">
+                        </div>
+                    </div>
+                </div>
+            </div>
+           <!--Week 9-->
+            <div class="labjournal-weekly">
+                <div>
+                    <ul class="nav nav-tabs">
+                      <li class="active"><a href="#a9" data-toggle="tab">Overview</a></li>
+                      <li><a href="#b9" data-toggle="tab">DelA-E</a></li>
+                    </ul>
+                </div>
+                <div class="jumbotron">
+                    <div class="scrollContent nav navbar" data-spy="scroll" data-target="#navbarExample" data-offset="0">
+                        <div class="tab-content">
+                             <div class="tab-pane active" id="a9">
                                  <p style="font-size:12pt; text-align:justify;">
-                                </html>{{:Team:Heidelberg/Templates/Del_week12_OP}}<html>
                                  </p>
                              </div>
-                             <div class="tab-pane" id="c12">
+                             <div class="tab-pane" id="b9">
                                  <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week12_FG}}<html>
+                                     </html>{{:Team:Heidelberg/Templates/Del_week10_AE}}<html>
                                  </p>
                              </div>
                          </div>
                      </div>
@@ Line 599: / Line 694: @@
              </div>
-                 <!--Week 13-->
+            <!--Week 10-->
              <div class="labjournal-weekly">
                  <div>
                      <ul class="nav nav-tabs">
-                       <li class="active"><a href="#a13" data-toggle="tab">Overview</a></li>
+                       <li class="active"><a href="#a10" data-toggle="tab">Overview</a></li>
-                       <li><a href="#b13" data-toggle="tab">DelA-E</a></li>
+                       <li><a href="#b10" data-toggle="tab">DelA-E</a></li>
-                       <li><a href="#c13" data-toggle="tab">DelA-F</a></li>
-                      <li><a href="#d13" data-toggle="tab">DelA-G</a></li>
-                      <li><a href="#h13" data-toggle="tab">DelE</a></li>
-                      <li><a href="#e13" data-toggle="tab">DelE-G</a></li>
-                      <li><a href="#j13" data-toggle="tab">DelF-G</a></li>
-                      <li><a href="#f13" data-toggle="tab">DelG</a></li>
-                      <li><a href="#g13" data-toggle="tab">DelO-P</a></li>
                      </ul>
                  </div>
-                 <div class="jumbotron weekly">
+                 <div class="jumbotron">
                      <div class="scrollContent nav navbar" data-spy="scroll" data-target="#navbarExample" data-offset="0">
-                         <div class="tab-content">
+                        <div class="tab-content">
-                             <div class="tab-pane active" id="a13">
+                             <div class="tab-pane active" id="a10">
                                  <p style="font-size:12pt; text-align:justify;">
                                  </p>
                              </div>
-                             <div class="tab-pane" id="b13">
+                             <div class="tab-pane" id="b10">
                                  <p style="font-size:12pt; text-align:justify;">
-                                      </html>{{:Team:Heidelberg/Templates/Del_week13_AE}}<html>
+                                      </html>{{:Team:Heidelberg/Templates/Del_week10_AE}}<html>
-                                </p>
-                            </div>
-                            <div class="tab-pane" id="c13">
-                                <p style="font-size:12pt; text-align:justify;">
-                                </html>{{:Team:Heidelberg/Templates/Del_week13_AF}}<html>
                                  </p>
                              </div>
-                            <div class="tab-pane" id="d13">
+                        </div>
-                                <p style="font-size:12pt; text-align:justify;">
+                    </div>
+                </div>
+            </div>
-                                </html>{{:Team:Heidelberg/Templates/Del_week13_AG}}<html>
+            <!--Week 11-->
+            <div class="labjournal-weekly">
+                <div>
+                    <ul class="nav nav-tabs">
+                      <li class="active"><a href="#a11" data-toggle="tab">Overview</a></li>
+                      <li><a href="#b11" data-toggle="tab">DelA-E</a></li>
+                    </ul>
+                </div>
+                <div class="jumbotron weekly">
+                    <div class="scrollContent nav navbar" data-spy="scroll" data-target="#navbarExample" data-offset="0">
+                       <div class="tab-content">
+                            <div class="tab-pane active" id="a11">
+                                <p style="font-size:12pt; text-align:justify;">
                                  </p>
                              </div>
-                             <div class="tab-pane" id="h13">
+                             <div class="tab-pane" id="b11">
                                  <p style="font-size:12pt; text-align:justify;">
-                                </html>{{:Team:Heidelberg/Templates/Del_week13_E}}<html>
+                                     </html>{{:Team:Heidelberg/Templates/Del_week11_AE}}<html>
                                  </p>
                              </div>
-                             <div class="tab-pane" id="e13">
+                       </div>
+                   </div>
+               </div>
+            </div>
+                    <!--Week 12-->
+            <div class="labjournal-weekly">
+                <div>
+                    <ul class="nav nav-tabs">
+                      <li class="active"><a href="#a12" data-toggle="tab">Overview</a></li>
+                      <li><a href="#b12" data-toggle="tab">DelA-E</a></li>
+                    </ul>
+                </div>
+                    <div class="jumbotron weekly">
+                    <div class="scrollContent nav navbar" data-spy="scroll" data-target="#navbarExample" data-offset="0">
+                        <div class="tab-content">
+                             <div class="tab-pane active" id="a12">
                                  <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week13_EG}}<html>
                                  </p>
                              </div>
-                             <div class="tab-pane" id="f13">
+                             <div class="tab-pane" id="b12">
                                  <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week13_G}}<html>
+                                     </html>{{:Team:Heidelberg/Templates/Del_week12_AE}}<html>
                                  </p>
                              </div>
-                             <div class="tab-pane" id="g13">
+                            <
+                        </div>
+                    </div>
+                </div>
+            </div>
+                 <!--Week 13-->
+            <div class="labjournal-weekly">
+                <div>
+                    <ul class="nav nav-tabs">
+                      <li class="active"><a href="#a13" data-toggle="tab">Overview</a></li>
+                      <li><a href="#b13" data-toggle="tab">DelA-E</a></li>
+                    </ul>
+                </div>
+                <div class="jumbotron weekly">
+                    <div class="scrollContent nav navbar" data-spy="scroll" data-target="#navbarExample" data-offset="0">
+                         <div class="tab-content">
+                             <div class="tab-pane active" id="a13">
                                  <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week13_OP}}<html>
                                  </p>
                              </div>
-                             <div class="tab-pane" id="j13">
+                             <div class="tab-pane" id="b13">
                                  <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week13_FG}}<html>
+                                     </html>{{:Team:Heidelberg/Templates/Del_week13_AE}}<html>
                                  </p>
                              </div>
                          </div>
@@ Line 683: / Line 831: @@
                        <li class="active"><a href="#a14" data-toggle="tab">Overview</a></li>
                        <li><a href="#b14" data-toggle="tab">DelA-E</a></li>
-                      <li><a href="#c14" data-toggle="tab">DelA-F</a></li>
-                      <li><a href="#d14" data-toggle="tab">DelA-G</a></li>
-                      <li><a href="#j14" data-toggle="tab">DelF-G</a></li>
-                      <li><a href="#f14" data-toggle="tab">DelG</a></li>
-                      <li><a href="#g14" data-toggle="tab">DelO-P</a></li>
                      </ul>
@@ Line 709: / Line 852: @@
                              </div>
-                            <div class="tab-pane" id="c14">
-                                <p style="font-size:12pt; text-align:justify;">
-                                </html>{{:Team:Heidelberg/Templates/Del_week14_AF}}<html>
-                                </p>
-                            </div>
-                            <div class="tab-pane" id="d14">
-                                <p style="font-size:12pt; text-align:justify;">
-                                </html>{{:Team:Heidelberg/Templates/Del_week14_AG}}<html>
-                                </p>
-                            </div>
-                            <div class="tab-pane" id="f14">
-                                <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week14_G}}<html>
-                                </p>
-                            </div>
-                            <div class="tab-pane" id="g14">
-                                <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week14_OP}}<html>
-                                </p>
-                            </div>
-                            <div class="tab-pane" id="j14">
-                                <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week14_FG}}<html>
-                                </p>
-                            </div>
                          </div>
@@ Line 751: / Line 865: @@
                        <li class="active"><a href="#a15" data-toggle="tab">Overview</a></li>
                        <li><a href="#b15" data-toggle="tab">DelA-E</a></li>
-                       <li><a href="#c15" data-toggle="tab">DelA-F</a></li>
-                      <li><a href="#d15" data-toggle="tab">DelA-G</a></li>
-                      <li><a href="#e15" data-toggle="tab">DelE-F</a></li>
-                      <li><a href="#j15" data-toggle="tab">DelF-G</a></li>
-                      <li><a href="#f15" data-toggle="tab">DelG</a></li>
-                      <li><a href="#g15" data-toggle="tab">DelO-P</a></li>
-                      <li><a href="#h15" data-toggle="tab">DelL</a></li>
-                      <li><a href="#i15" data-toggle="tab">pSB4K5</a></li>
                      </ul>
                  </div>
@@ Line 779: / Line 886: @@
-                            <div class="tab-pane" id="c15">
-                                <p style="font-size:12pt; text-align:justify;">
-                                     </html>{{:Team:Heidelberg/Templates/Del_week15_AF}}<html>
-                                </p>
-                            </div>
-                            <div class="tab-pane" id="d15">
+                       </div>
-                                <p style="font-size:12pt; text-align:justify;">
+                   </div>
+               </div>
+            </div>
-                                </html>{{:Team:Heidelberg/Templates/Del_week15_AG}}<html>
+             <!--Week 16-->
+            <div class="labjournal-weekly">
+                <div>
+                    <ul class="nav nav-tabs">
+                      <li class="active"><a href="#a16" data-toggle="tab">Overview</a></li>
+                      <li><a href="#b16" data-toggle="tab">DelA-F</a></li>
+                    </ul>
+                </div>
+                <div class="jumbotron weekly">
+                    <div class="scrollContent nav navbar" data-spy="scroll" data-target="#navbarExample" data-offset="0">
+                       <div class="tab-content">
+                            <div class="tab-pane active" id="a16">
+                                <p style="font-size:12pt; text-align:justify;">
                                  </p>
                              </div>
+                             <div class="tab-pane" id="b16">
-                             <div class="tab-pane" id="e15">
                                  <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week15_EF}}<html>
+                                     </html>{{:Team:Heidelberg/Templates/Del_week16_AF}}<html>
                                  </p>
                              </div>
-                             <div class="tab-pane" id="f15">
-                                <p style="font-size:12pt; text-align:justify;">
+                       </div>
-                               </html>{{:Team:Heidelberg/Templates/Del_week15_G}}<html>
+                   </div>
-                                </p>
+               </div>
-                            </div>
+            </div>
-                             <div class="tab-pane" id="g15">
+            <!--Week 17-->
+            <div class="labjournal-weekly">
+                <div>
+                    <ul class="nav nav-tabs">
+                      <li class="active"><a href="#a17" data-toggle="tab">Overview</a></li>
+                      <li><a href="#i17" data-toggle="tab">pSB4K5</a></li>
+                    </ul>
+                </div>
+                <div class="jumbotron weekly">
+                    <div class="scrollContent nav navbar" data-spy="scroll" data-target="#navbarExample" data-offset="0">
+                       <div class="tab-content">
+                             <div class="tab-pane active" id="a17">
                                  <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week15_OP}}<html>
                                  </p>
                              </div>
-                             <div class="tab-pane" id="h15">
+                             <div class="tab-pane" id="i17">
                                  <p style="font-size:12pt; text-align:justify;">
-                                </html>{{:Team:Heidelberg/Templates/Del_week15_L}}<html>
+                                </html>{{:Team:Heidelberg/Templates/Del_week17_pSB4K5}}<html>
                                  </p>
                              </div>
-                             <div class="tab-pane" id="i15">
+                       </div>
+                   </div>
+               </div>
+            </div>
+            <!--Week 18-->
+            <div class="labjournal-weekly">
+                <div>
+                    <ul class="nav nav-tabs">
+                      <li class="active"><a href="#a18" data-toggle="tab">Overview</a></li>
+                      <li><a href="#i18" data-toggle="tab">pSB4K5</a></li>
+                    </ul>
+                </div>
+                <div class="jumbotron weekly">
+                    <div class="scrollContent nav navbar" data-spy="scroll" data-target="#navbarExample" data-offset="0">
+                       <div class="tab-content">
+                             <div class="tab-pane active" id="a18">
                                  <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week15_pSB4K5}}<html>
                                  </p>
                              </div>
-                             <div class="tab-pane" id="j15">
+                             <div class="tab-pane" id="i18">
                                  <p style="font-size:12pt; text-align:justify;">
-                                </html>{{:Team:Heidelberg/Templates/Del_week15_FG}}<html>
+                                </html>{{:Team:Heidelberg/Templates/Del_week17_pSB4K5}}<html>
                                  </p>
                              </div>
                         </div>
@@ Line 834: / Line 992: @@
              </div>
-             <!--Week 16-->
+           <!--Week 19-->
              <div class="labjournal-weekly">
                  <div>
                      <ul class="nav nav-tabs">
-                       <li class="active"><a href="#a16" data-toggle="tab">Overview</a></li>
+                       <li class="active"><a href="#a19" data-toggle="tab">Overview</a></li>
-                       <li><a href="#b16" data-toggle="tab">DelA-F</a></li>
+                       <li><a href="#i19" data-toggle="tab">pSB4K5</a></li>
-                      <li><a href="#d16" data-toggle="tab">DelA-G</a></li>
-                      <li><a href="#j16" data-toggle="tab">DelF-G</a></li>
-                      <li><a href="#f16" data-toggle="tab">DelG</a></li>
-                      <li><a href="#g16" data-toggle="tab">DelO-P</a></li>
-                      <li><a href="#i16" data-toggle="tab">pSB4K5</a></li>
-                       <li><a href="#z16" data-toggle="tab">Gibson assembly and validation</a></li>
                      </ul>
                  </div>
@@ Line 852: / Line 1,005: @@
                         <div class="tab-content">
-                             <div class="tab-pane active" id="a16">
+                             <div class="tab-pane active" id="a19">
                                  <p style="font-size:12pt; text-align:justify;">
@@ Line 858: / Line 1,011: @@
                              </div>
-                             <div class="tab-pane" id="b16">
+                             <div class="tab-pane" id="i19">
                                  <p style="font-size:12pt; text-align:justify;">
-                                     </html>{{:Team:Heidelberg/Templates/Del_week16_AF}}<html>
+                               </html>{{:Team:Heidelberg/Templates/Del_week17_pSB4K5}}<html>
                                  </p>
                              </div>
-                             <div class="tab-pane" id="d16">
-                                <p style="font-size:12pt; text-align:justify;">
-                                </html>{{:Team:Heidelberg/Templates/Del_week16_AG}}<html>
+                       </div>
+                   </div>
+               </div>
+            </div>
+           <!--Week 20-->
+            <div class="labjournal-weekly">
+                <div>
+                    <ul class="nav nav-tabs">
+                      <li class="active"><a href="#a20" data-toggle="tab">Overview</a></li>
+                      <li><a href="#i20" data-toggle="tab">pSB4K5</a></li>
+                    </ul>
+                </div>
+                <div class="jumbotron weekly">
+                    <div class="scrollContent nav navbar" data-spy="scroll" data-target="#navbarExample" data-offset="0">
+                       <div class="tab-content">
+                            <div class="tab-pane active" id="a20">
+                                <p style="font-size:12pt; text-align:justify;">
                                  </p>
                              </div>
-                             <div class="tab-pane" id="f16">
+                             <div class="tab-pane" id="i20">
                                  <p style="font-size:12pt; text-align:justify;">
-                                </html>{{:Team:Heidelberg/Templates/Del_week16_G}}<html>
+                                </html>{{:Team:Heidelberg/Templates/Del_week17_pSB4K5}}<html>
                                  </p>
                              </div>
-                             <div class="tab-pane" id="g16">
+                       </div>
+                   </div>
+               </div>
+            </div>
+           <!--Week 21-->
+            <div class="labjournal-weekly">
+                <div>
+                    <ul class="nav nav-tabs">
+                      <li class="active"><a href="#a21" data-toggle="tab">Overview</a></li>
+                      <li><a href="#i21" data-toggle="tab">pSB4K5</a></li>
+                    </ul>
+                </div>
+                <div class="jumbotron weekly">
+                    <div class="scrollContent nav navbar" data-spy="scroll" data-target="#navbarExample" data-offset="0">
+                       <div class="tab-content">
+                             <div class="tab-pane active" id="a21">
                                  <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week16_OP}}<html>
                                  </p>
                              </div>
-                             <div class="tab-pane" id="i16">
+                             <div class="tab-pane" id="i21">
                                  <p style="font-size:12pt; text-align:justify;">
-                                </html>{{:Team:Heidelberg/Templates/Del_week16_pSB4K5}}<html>
+                                </html>{{:Team:Heidelberg/Templates/Del_week17_pSB4K5}}<html>
                                  </p>
                              </div>
-                             <div class="tab-pane" id="j16">
+                       </div>
+                   </div>
+               </div>
+            </div>
+             <!--Week 22-->
+            <div class="labjournal-weekly">
+                <div>
+                    <ul class="nav nav-tabs">
+                      <li class="active"><a href="#a22" data-toggle="tab">Overview</a></li>
+                      <li><a href="#i22" data-toggle="tab">pSB4K5</a></li>
+                    </ul>
+                </div>
+                <div class="jumbotron weekly">
+                    <div class="scrollContent nav navbar" data-spy="scroll" data-target="#navbarExample" data-offset="0">
+                       <div class="tab-content">
+                             <div class="tab-pane active" id="a22">
                                  <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Templates/Del_week16_FG}}<html>
                                  </p>
                              </div>
-                             <div class="tab-pane" id="z16">
+                             <div class="tab-pane" id="i22">
                                  <p style="font-size:12pt; text-align:justify;">
-                                </html>{{:Team:Heidelberg/Template/Del_week16_Gibson}}<html>
+                                </html>{{:Team:Heidelberg/Templates/Del_week17_pSB4K5}}<html>
                                  </p>
                              </div>
                         </div>
@@ Line 910: / Line 1,128: @@
              </div>
-            <!--Week 17-->
+           <!--Week 23-->
              <div class="labjournal-weekly">
                  <div>
                      <ul class="nav nav-tabs">
-                       <li class="active"><a href="#a17" data-toggle="tab">Overview</a></li>
+                       <li class="active"><a href="#a23" data-toggle="tab">Overview</a></li>
-                       <li><a href="#i17" data-toggle="tab">pSB4K5</a></li>
+                       <li><a href="#i23" data-toggle="tab">pSB4K5</a></li>
-                       <li><a href="#z17" data-toggle="tab">Gibson assembly and validation</a></li>
                      </ul>
                  </div>
@@ Line 923: / Line 1,141: @@
                         <div class="tab-content">
-                             <div class="tab-pane active" id="a17">
+                             <div class="tab-pane active" id="a237">
                                  <p style="font-size:12pt; text-align:justify;">
@@ Line 931: / Line 1,149: @@
-                             <div class="tab-pane" id="i17">
+                             <div class="tab-pane" id="i23">
                                  <p style="font-size:12pt; text-align:justify;">
                                 </html>{{:Team:Heidelberg/Templates/Del_week17_pSB4K5}}<html>
@@ Line 937: / Line 1,155: @@
                              </div>
-                             <div class="tab-pane" id="z17">
-                                <p style="font-size:12pt; text-align:justify;">
-                               </html>{{:Team:Heidelberg/Template/Del_week17_Gibson}}<html>
-                                </p>
-                            </div>
                         </div>

Team:Heidelberg/Delftibactin/Delftibactin

From 2013.igem.org

Revision as of 08:39, 4 October 2013

Team

Project

Notebook

Parts

Software

Human Practice

Safety

Modeling

Delftibactin. Bringing it all together.

Week 1

Week 2

Week 3

Week 4

Week 5

Week 6

Week 7

Week 8

Week 9

Week 10

Week 11

Week 12

Week 13

Week 14

Week 15

Week 16

Week 17

Week 18

Week 19

Week 20

Week 21

Week 22

Week 23

Methods:

06-07-2013

Amplification from FS_02 to FS_03; 5.3 kb

03-07-2013

Amplification from FS_02 to FS_05; 11.2 kb

04-07-2013

Amplification from FS_02 to FS_05; 11.2 kb

05-08-2013

Amplification from FS_02 to FS_07; 16.4 kb

06-07-2013

Amplification from FS_04 to FS_07; 11.1 kb

03-07-2013

Amplification from FS_06 to FS_09; 8.5 kb

Amplification from FS_06 to FS_09; 8.5 kb

Amplification from FS_06 to FS_11; 11.6 kb

05-07-2013

Amplification from FS_06 to FS_07; 5.2 kb

05-07-2013

Amplification from FS_08 to FS_11; 6.5 kb

07-07-2013

Amplification from FS_08 to FS_11; 6.5 kb

06-07-2013

Amplification from FS_02 to FS_03; 5.3 kb

06-07-2013

Amplification from FS_02 to FS_03; 5.3 kb

03-07-2013

Amplification from FS_02 to FS_05; 11.2 kb

04-07-2013

Amplification from FS_02 to FS_05; 11.2 kb

06-07-2013

Amplification from FS_02 to FS_03; 5.3 kb

06-07-2013

Amplification from FS_02 to FS_03; 5.3 kb

06-07-2013

Amplification from FS_02 to FS_03; 5.3 kb

06-07-2013

Amplification from FS_02 to FS_03; 5.3 kb

06-07-2013

Amplification from FS_02 to FS_03; 5.3 kb

06-07-2013

Amplification from FS_02 to FS_03; 5.3 kb

06-07-2013

Amplification from FS_02 to FS_03; 5.3 kb

08-07-2013

Amplification from FS_02 to FS_03; 5.3 kb