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Pathway Notebook

Week	Days
	Monday	Tuesday	Wednesday	Thursday	Friday	Saturday	Sunday
31	8-02-2010	8-03-2010	8-04-2010	8-05-2010	8-06-2010	8-07-2010	8-08-2010
32	8-09-2010	8-10-2010	8-11-2010	8-12-2010	8-13-2010	8-14-2010	8-15-2010
33	8-16-2010	8-17-2010	8-18-2010	8-19-2010	8-20-2010	8-21-2010	8-22-2010
34	8-23-2010	8-24-2010	8-25-2010	8-26-2010	8-27-2010	8-28-2010	8-29-2010
35	8-30-2010	8-31-2010	9-01-2010	9-02-2010	9-03-2010	9-04-2010	9-05-2010
36	9-06-2010	9-07-2010	9-08-2010	9-09-2010	9-10-2010	9-11-2010	9-12-2010
37	9-13-2010	9-14-2010	9-15-2010	9-16-2010	9-17-2010	9-18-2010	9-19-2010
38	9-20-2010	9-21-2010	9-22-2010	9-23-2010	9-24-2010	9-25-2010	9-26-2010
39	9-27-2010	9-28-2010	9-29-2010	9-30-2010	10-01-2010	10-02-2010	10-03-2010
40	10-04-2010	10-05-2010	10-06-2010	10-07-2010	10-08-2010	10-09-2010	10-10-2010
41	10-11-2010	10-12-2010	10-13-2010	10-14-2010	10-15-2010	10-16-2010	10-17-2010
42	10-18-2010	10-19-2010	10-20-2010	10-21-2010	10-22-2010	10-23-2010	10-24-2010
43	10-25-2010	10-26-2010	10-27-2010	10-28-2010	10-29-2010	10-30-2010	10-31-2010

8-02-2010

Some test text here.

8-03-2010

Some test text in bold We created following tests:

test1
test2
test3

8-04-2010

Example of a table

header 1	header 2	header 3
row 1, cell 1	row 1, cell 2	row 1, cell 3
row 2, cell 1	row 2, cell 2	row 2, cell 3

8-05-2010

gDNA prep (cultures from 7-28-2010) with innuPREP Bacteria DNA kit

-> additionally TE-buffer (100µl EDTA [o.5M]; 500µl Tris [1M]) was made, pH=8.0

8-06-2010

text

8-07-2010

weekend

8-08-2010

weekend

8-09-2010

PCR were performed as follows:

mastermix:

MQ: 93.6µl

10xbuffer: 12.0µl

dNTP's: 2.4µl (each 10mM)

Phusion: 1.2µl (Pfu-Promega)

->109.2µl/6 = 18.2µl each tube

	1	2	3	4	5	6
primer fwd	pduAfwd (1)	pduJfwd (7)	1P1D (12)	mpduDfwd (9)	5P3AD (14)	5P3AD (14)
primer rev	pduJrev (8)	pduUrev (2)	mpduDrev (10)	3P2D (13)	9P4A (15)	9P4A (15)
template	Knut	Knut	gDNA citrobacter freundii	gDNA c. freundii	gDNA streptomyces thioluteus, glycerolstock	gDNA s. thioluteus, LB prep

-> no products on gel picture

8-10-2010

PCR were performed with pdu-template:

mastermix:

primer fwd/rev: 1µl

MQ: 15.88µl

10xbuffer: 5µl

DMSO: 0.625µl

dNTP's: 0.5µl (each 10mM)

Phusion: 0.4µl (Pfu-Promega)

25µl

->109.2µl/6 = 18.2µl each tube

this mix was produced for all of the six primer pairs (see 9.8.10)

->again no spcific product was seen on the gel picture

8-11-2010

text

8-12-2010

text

8-13-2010

text

8-14-2010

weekend

8-15-2010

weekend

8-16-2010

text

8-17-2010

PCR mastermix

MQ: 34.4µl

10xbuffer: 5µl

pF: 3µl

pR: 3µl

dNTP's: 2µl

Pfu(GeneON): 0.6µl

-> 50µl

primer combination1: 1+8 (pduA+mpduJ)
primer combination2: 2+7 (mpduJ+pduU)

8-18-2010

text

8-19-2010

PCR:

charge	1	2	3
MQ [µl]	13.2	11.7	8.7
10xbuffer [µl]	2.5	2.5	2.5
DMSO [µl]	0.625	0.625	0.625
pF [µl]	3	3	3
pR [µl]	3	3	3
dNTP's [µl]	2	2	2
template [ng/µl]	0.5	2	5
Pfu [µl]	0.5	0.5	0.5

-> each 25µl

primer combination1: 1+8 (Knut [5ng;1ng])
primer combination2: 1+6 (Knut [6ng;2ng])
primer combination3: 2+5 (Knut [7ng;3ng])
primer combination4: 2+7 (Knut [8ng;4ng])

transformation efficiency from competent cells: 5.6x10⁶

8-20-2010

PCR mastermix

MQ: 24.75µl >-	DMSO: 1.25µl
10xbuffer: 5µl
pF: 3µl
pR: 3µl
template: 10µl
dNTP's: 2µl
Pfu(GeneON): 1µl

-> 50µl

primer combination1: 1+6
primer combination2: 2+5

8-21-2010

weekend

8-22-2010

weekend

8-23-2010

text

8-24-2010

Preparing Competent Cells with Benny's protocoll & buffers

- grow the culture to an OD ₆₀₀ of ~ 0,3

-> our cells: OD ₆₀₀ = 0,256

- centrifuge for 10 min at 4°C at 3000rpm

- resuspend the pellet in buffer 1

-> we used 400 µl

- centrifuge for 10 min at 4°C at 2500rpm

- resuspend the pellet in less buffer 1 than before

-> we used 200 µl

- add buffer 2 in a ratio of 1:10 = buffer 2:buffer 1

-> we added 20µl of buffer 2

- incubate on ice for 10 min

-> as we probably used too little buffer, we added another 200 µl of buffer 1 and 20µl of buffer 2

- aliquote the suspension and shockfreeze it at -80°C

-> we put 50 µl in each eppendorf

Test-Transformation in order to see whether the cells will work

- done with protocoll (3 Transformation)

-> we tested with the following DNA:

1. K098200 (biobrick) [Amp., 5 µl]

2. pUC [Amp., 1µl]

-> as the cells are probably in a to high concentration, we thined them down with saline to an end-concentration of 10 ^-2

8-25-2010

again: Preparing Competent Cells with Benny's protocoll & buffers

- grow the culture to an OD ₆₀₀ of ~ 0,3

-> our cells: OD ₆₀₀ = 0,22

- centrifuge for 10 min at 4°C at 3000rpm

- resuspend the pellet in buffer 1

-> we used 16 ml

- centrifuge for 10 min at 4°C at 2500rpm

- resuspend the pellet in less buffer 1 than before

-> we used 2 ml

- then we allocated the suspension into two eppendorfs

- in eppendorf + we added buffer 2 in a ratio of 1:20 = buffer 2:buffer 1

-> we added 50µl of buffer 2

- incubate on ice for 10 min

- aliquote the suspension and shockfreeze it at -80°C

-> we put 50 µl in each eppendorf

Test-Transformation in order to see whether the cells will work

- done with protocoll (3 Transformation)

-> we tested with the following DNA: pUC (10pn/µl, Amp) 1 µl was used

therefore we made fresh pUC (10pn/µl): 0,5 µl pUC-stock (0,5µg/µl) + 25 ml H₂O)

8-26-2010

joining PCR

* hotstart

MQ: 11.05µl

hotstart: 25µl

MgCl: 5µl

primer forw: 3µl (2-6)

primer rev: 3µl (1-5)

25µl

* Extender

MQ: 37.75µl

Puffer10x: 5.4µl

DMSO: 1.25µl

primer forw: 2µl (1-5)

primer rev: 2µl (2-6)

dNTP's: 2µl

polymerase: 1µl

54µl

PCR-programm:

94°C 1'

94°C 30
39°C (hot1/ex1) / 44.2°C (hot2/ex2) / 50.5°C (hot3/ex3) / 56.7°C (hot4/ex4) / 65°C (hot5/ex5)
73°C 3' (elongationtime/cycle plus 10 sec)

73°C 1'

8-27-2010

text

8-28-2010

weekend

8-29-2010

weekend

8-30-2010

text

8-31-2010

1: PCR- pfu (Promega)

MQ: 24.75µl

DMSO: 1.25µl

buffer10x: 5µl

primer fwd: 3µl

primer rev: 3µl

dNTP's: 2µl

polymerase pfu: 1µl

=> 50µl

PCR-programm:

1: 94°C 1'

2: 94°C 30"
3: 52°C 30"
4: 73°C 3'
5: repeat step 2-4 35x

6: 73°C 3'

=> no fragments

2: PCR- DreamTaq Green

MQ: 25.5µl

DMSO: 1µl

buffer10x: 5µl

primer fwd: 3µl (#1)

primer rev: 3µl (#2)

dNTP's: 2µl

template[ng/µl]: 10µl

polymerase DreamTaq: 0.5µl

=> 50µl

PCR-programm:

1: 95°C 2'

2: 95°C 30"
3: 52°C 30"
4: 72°C 2'30"
5: repeat step 2-4 30x

6: 72°C 5'

=> no fragments with right size

3: JoiningPCR- pdu

primer combinations: 1+6 & 2+5

in the same way as PCR1&2

=> no fragments

9-01-2010

1: PCR- amplification of the pdu-operon

primer 5+8 => testing the template; 2 equal charges to check template

PCRmastermix(2x): 10µl

primer fwd: 1.5µl

primer rev: 1.5µl

DMSO: 0.5µl

MQ: 4.5µl / 2.5µl

each charge 20µl

PCR-programm:

1: 95°C 2'

2: 95°C 30"
3: 50°C 30"
4: 72°C 2'30"
5: repeat step 2-4 35x

6: 72°C 5'

2: testing the plasmid via gelelectrophoresis
3: PCR- amplification of AurF (from streptomyces thioluteus)

PCRmastermix(2x): 10µl

primer fwd: 1.5µl

primer rev: 1.5µl

DMSO: 0.5µl

MQ: 1.5µl

=> 20µl

PCR-programm:

1: 95°C 2'

2: 95°C 30"
3: 54°C 30"
4: 72°C 2'30"
5: repeat step 2-4 35x

6: 72°C 5'

9-02-2010

PCR1- pduB->pduU

primer2+5

	PCR mastermix(2x)(+Taq)	primer fwd	primer rev	template [0.5µg/ml]	MQ	DMSO
a)	10µl	1.5µl	1.5µl	4µl	2.5µl	0.5µl
b)	10µl	1.5µl	1.5µl	2µl	4.5µl	0.5µl

each charge 20µl

PCR-programm:

1: 95°C 2'

2: 95°C 30"
3: 50°C 30"
4: 72°C 2'30"
5: repeat step 2-4 35x

6: 72°C 5'

PCR2- amplification of pdu

PCRmastermix: 10µl (Taq-polymerase)

primer fwd: 1.5µl

primer rev: 1.5µl

template[ng/µl]: 4µl

MQ: 2.5µl

DMSO: 0.5µl

each charge 20µl

primer combinations:

1: 1+4
2: 3+6
3: 5+8
4: 7+2

PCR-programm:

1: 95°C 2'

2: 95°C 30"
3: 50°C 30"
4: 72°C 3'
5: repeat step 2-4 35x

6: 72°C 5'

PCR3- amplification of pdu

buffer5x: 20µl (Taq-polymerase)

primer fwd: 5µl

primer rev: 5µl

template[ng/µl]: 10µl

MQ: 51.5µl

DMSO: 2.5µl

dNTP's: 5µl

Phusion polymerase: 1µl

100µl => each charge 25µl

primer combinations:

1: 1+4
2: 3+6
3: 5+8
4: 7+2

PCR-programm:

1: 98°C 1'

2: 98°C 30"
3: 50°C 30"
4: 72°C 2'30"
5: repeat step 2-4 35x

6: 72°C 5'

9-03-2010

PCR- amplification of pdu primer 5+8

MQ: 51.5µl

buffer5x: 20µl

primer fwd: 5µl

primer rev: 5µl

dNTP's: 5µl

DMSO: 2.5µl

Phusion polymerase: 1µl

100µl => 5 charges, each 20µl

PCR-programm with temperature gradient:

1: 98°C 30"

2: 98°C 10"
3: Tx 30"
4: 72°C 1'
5: repeat step 2-4 30x

6: 72°C 5'

charge	1	2	3	4	5
Tx [°C]	50	52.5	56.4	61	64.7

9-04-2010

weekend

9-05-2010

weekend

9-06-2010

PCRamplification of pdu

MQ	51.5µl
buffer5x	20µl
primer fwd	5µl
primer rev	5µl
dNTP's	5µl
DMSO	2.5µl
template	10µl
Phusion polymerase	1µl
sum	100µl

=> 5charges, each 20µl

*primercombination 1: 1+4

*primercombination 2: 3+6

*primercombination 3: 5+8

*primercombination 4: 7+2

PCRprogramm:

1:98°C 30"

2: 98°C 10"

3: 54°C 30"

4: 72°C 1'

5: repeat 2-4 30x

6: 72°C 5'

7: 15°C break

=> no fragmentes on gel

PCRamplification of pduD

PCRmastermix	10µl
primer fwd	1.5µl
primer rev	1.5µl
template	4µl
DMSO	0.5µl
MQ	2.5µl
sum	20µl

PCRprogramm

1: 94°C 2'

2: 94°C 30"

3: 50°C 30"

4: 72°C 2'

5: repeat 2-4 30x

6: 72°C 10'

7: 15°C break

*primercombination 1: 12+10

*primercombination 2: 9+13

PCRamplification of pdu

MQ	51.5µl
buffer5x	20µl
primer fwd	5µl
primer rev	5µl
dNTP's	5µl
DMSO	2.5µl
template	10µl
Phusion polymerase	1µl
sum	100µl

=> 5charges, each 20µl

*primercombination 1: 1+4

9-07-2010

PCRamplification of pduD

with PCR mastermix

PCRmastermix	50µl
primer fwd	7.5µl
primer rev	7.5µl
template	20µl
DMSO	2.5µl
MQ	12.5µl
sum	100µl

=> no product on gel

9-08-2010

PCRamplification of pdu-operon

with PCR mastermix

PCRmastermix	10µl
primer fwd	1.5µl
primer rev	1.5µl
template	2µl
DMSO	0.5µl
MQ	4.5µl
sum	20µl

PCR programm

1: 94°C 2'

2: 94°C 30"

3: 50°C 30"

4: 72°C 2'

5: repeat 2-4 30x

6: 72°C 10'

7: 15°C break

with Pfu polymerase

MQ	45µl
buffer	8µ
primer fwd	6µl
primer rev	6µl
template	8µl
dNTP's	4µl
DMSO	2µl
Pfu polymerase	1µl
sum	80µ

=>4 charges, each 20µl

*primercombination 1: 1+4

*primercombination 2: 3+6

*primercombination 3: 5+8

*primercombination 4: 7+2

PCRprogramm as before

9-09-2010

PCR amplification of pduD from Citrobacter freundii

buffer10x: 2µl
primer fwd: 1.5µl
primer rev: 1.5µl
template: 2µl
DMSO: 0.5µl
MQ: 11.25µl
dNTP's: 1µl
PCRextender polymerase: 1µl
sum	20µl

primer combination 1: #1 #4
primer combination 2: #3 #6
primer combination 3: #5 #8
primer combination 4: #7 #2

PCR programm for charge 1&2:

1: 94°C 2'

2: 94°C 20"

3: 50°C 20"

4: 72°C 40"

5: go to 2; 35x

6: 72°C 10'

PCR programm for charge 3&4:

1: 94°C 2'

2: 94°C 20"

3: 52°C 20"

4: 72°C 1'30"

5: go to 2; 35x

6: 72°C 10'

PCRamplification of pduD

charge	1	3	2	4
PCRmastermix	10	10	10	10
primer fwd	1.5µl	1.5µl	1.5µl	1.5µl
primer rev	1.5µl	1.5µl	1.5µl	1.5µl
template	2	2	4	4
DMSO	0.5µl	0.5µl	0.5µl	0.5µl
MQ	4.5µl	4.5µl	2.5µl	2.5µl
sum	20µl	20µl	20µl	20µl

primer combination 1&2: #1 #4
primer combination 3&4: #3 #6

PCR programm

1: 94°C 2'

2: 94°C 20"

3: 52°C 20"

4: 72°C x[t]

5: go to 2; 35x

6: 72°C 10'

charge	1	2	3	4
x[t]	40"	40"	1'30"	1'30"

9-10-2010

PCR amplification of pduD from Citrobacter freundii

PCRmastermix: 10µl (+Taq)
primer fwd: 1.5µl
primer rev: 1.5µl
template: 2µl
DMSO: 0.5µl
MQ: 4.5µl
sum	20µl

primer combination 1: #12 #15
primer combination 2: #12 #13
primer combination 3: #14 #15

PCR programm:

1: 94°C 2'

2: 94°C 30"

3: 52°C 30"

4: 72°C 2'

5: go to 2; 35x

6: 72°C 10'

7: 15°C 5'

=> no result

testing transformation with Sina-Science-Services compis(250µl) and knut plasmid(10µl; 0.5µg/µl)

transformation as protocol from the manufacturer...
5µl (1:40 diluted) on CAM- and AMP-plate
rest of them(~100µl) preculture, then 1l culture with LB (CAM/AMP)

9-11-2010

weekend

9-12-2010

weekend

9-13-2010

touchdown PCR of pduD

1: 94°C 2'

2: 94°C 30"

3: 58°C/56°C/54°C/52°C/50°C/48° 30"

4: 72°C 2'

5: (for each temperature)repeat 2-4 2x

6: 94°C 30"

7: 52°C 30"

8: 72°C 2'

9: repeat 6-8 29x

10: 72°C 10'

11: 15°C break

9-14-2010

PCR extender

MQ	2x57µl
buffer	2x10µl
primer fwd	2x7.5µl
primer rev	2x7.5µl
template	2x10µl
dNTP's	2x4.5µl
DMSO	2x2.5µl
polymerase	2x1µl

2x100µl => 4charges each 50µl

primer combination as yesterday

9-15-2010

control-gel of fragment 2, 3 (->15ng), 4 (->20mg) GELFOTO REINSTELLEN

joining PCR-pduOperon with fragments 3 & 4 mit PCR Extender

buffer: 5µl

MQ: 26,5

primer: 2*2µl

fragment 4: 5µl

fragmet 3: 4,5 µl

dNTPs: 3µl

DMSO: 1,5µl

PCR Extender Pol.: o,5µl

sum: 50µl

PCR-program:

94°C 2min

94°C 20sec

52°C 20sec

72°C 5min

repeat these steps 35x

72°C 10min

GELFOTO REINSTELLEN

9-16-2010

joining PCR-pduOperon with fragments 3 & 4 mit PCR Extender, primer were added later

charge: same as yesterday

pcr-program:

first without primer:

94°C 2min

94°C 20sec

52°C 20sec

72°C 5min

repeat these steps 10x

72°C 10min

then with primer:

94°C 2min

94°C 20sec

52°C 20sec

72°C 5min

repeat these steps 30x

72°C 10min

GELFOTO REINSTELLEN

->reproducable results

control-gel and eluation of the joining-fragments

GELFOTO REINSTELLEN

purification of pdu-fragment1

precipitation with EtOH + Na-Acetat (pH5,2)

precipitation of DNA-fragments <200bp

1 vol. DNA (z.B. 100µl)

1/10 vol. 3M Na-Acetat pH5,2

1µl Glycogen (optional)

2 vol. 100% EtPH, -20°C

-> -20°C, 30min

-> max rpm, 15min

->discard supernatant (with pipett)

->"flick-spin"

->discard the rest of the supernatant

->dry at room temperature 5-10min

->resolve in MQ

9-17-2010

joining-PCR of the pduOperon fragment1&2

for 100µl joining-template with PCRextender

Buffer 5x	5µl
H₂O	27.75µl
dNTPs	3µl
Primer 1	2µl
Primer 6	2µl
DMSO	1,5µl
template 1	1.25µl
template 2	7µl
PCRextender polymerase	0,5µl
sum	50µl

PCR programm

without primer

94°C 2'

94°C 20"

52°C 20"

72°C 5'

repeat 30x

72°C 10'

with primer

94°C 2'

94°C 20"

52°C 20"

72°C 5'

repeat 30x

72°C 10'

PCRamplification of pduD

with PCRmastermix

template	2µl
MasterMix for Taq	10µl
Primer *2	1.5µl *2
DMSO	0,5µl
H₂O	4.5µl
sum	20µl

PCR-programm

94°C 2'

94°C 20"

52°C 30"

72°C 3'

repeat 30x

72°C 10'

GELPHOTO REINSTELLEN

9-18-2010

weekend

9-19-2010

weekend

9-20-2010

1. PCRamplification of pduD with PCRextender

buffer	5µl
primer fwd	2µl
primer rev	2µl
template	5µl
dNTP's	3µl
DMSO	1.5µl
MQ	31µl
PCRextender polymerase	0.5µl
sum	50µl

PCRprogramm

94°C 2'

94°C 20"

52°C 20"

72°C 3'

repeat 30x

72°C 10'

=> no result

2. purification of joining-product 1/2 from yesterday

3. reamplification of joining-product 3/4

with PCRextender

buffer	5µl
primer fwd	2µl
primer rev	2µl
template	5µl
dNTP's	3µl
DMSO	1.5µl
MQ	31µl
PCRextender polymerase	0.5µl
sum	50µl

PCRprogramm

94°C 2'

94°C 20"

52°C 30"

72°C 5'

repeat 30x

72°C 10'

9-21-2010

test PCRamplification of pdu-operon (fragment1/2) with PCRextender

buffer	5µl
primer fwd	2µl
primer rev	2µl
template	4µl
dNTP's	3µl
DMSO	1.5µl
MQ	32µl
PCRextender polymerase	0.5µl
sum	50µl

PCRprogramm

94°C 2'

94°C 20"

52°C 20"

72°C 2'

repeat 35x

72°C 10'

test PCRamplification of pdu-operon (fragment1/2) with PCRextender

buffer	5µl
primer fwd	2µl
primer rev	2µl
template	4µl
dNTP's	3µl
DMSO	1.5µl
MQ	32µl
PCRextender polymerase	0.5µl
sum	50µl

PCRprogramm

94°C 2'

94°C 20"

52°C 20"

72°C 2'

repeat 35x

72°C 10'

test PCRamplification of pdu-operon (fragment1/2) with PCRextender

{|

|primer fwd |1.5µl |- |primer rev |1.5µl |- |template (gDNA streptomyces thioluteus) |2µl |- |DMSO |0.5µl |- |MQ |4.5µl |- |PCRmastermix |10µl |- |sum |20µl |}

=> 2 charges each 10µl

similar charge for colonyPCR

PCRprogramm

94°C 2'

94°C 30"

52°C 30"

72°C 2'

repeat 30x

72°C 5'

9-22-2010

text

9-23-2010

text

9-24-2010

text

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@@ Line 1,775: / Line 1,775: @@
 |-
 |72°C 10'
+|}
+<font color="#009933">test PCRamplification of pdu-operon</font> (fragment1/2)
+with PCRextender
+::{|
+|buffer
+|5µl
+|-
+|primer fwd
+|2µl
+|-
+|primer rev
+|2µl
+|-
+|template
+|4µl
+|-
+|dNTP's
+|3µl
+|-
+|DMSO
+|1.5µl
+|-
+|MQ
+|32µl
+|-
+|PCRextender polymerase
+|0.5µl
+|-
+|sum
+|50µl
+|}
+PCRprogramm
+::{|
+|94°C 2'
+----
+|-
+|94°C 20"
+|-
+|52°C 20"
+|-
+|72°C 2'
+|-
+|repeat 35x
+----
+|-
+|72°C 10'
+|}
+<font color="#009933">test PCRamplification of pdu-operon</font> (fragment1/2)
+with PCRextender
+*::{|
+|primer fwd
+|1.5µl
+|-
+|primer rev
+|1.5µl
+|-
+|template (gDNA ''streptomyces thioluteus'')
+|2µl
+|-
+|DMSO
+|0.5µl
+|-
+|MQ
+|4.5µl
+|-
+|PCRmastermix
+|10µl
+|-
+|sum
+|20µl
+|}
+=> 2 charges each 10µl
+*similar charge for colonyPCR
+PCRprogramm
+::{|
+|94°C 2'
+----
+|-
+|94°C 30"
+|-
+|52°C 30"
+|-
+|72°C 2'
+|-
+|repeat 30x
+----
+|-
+|72°C 5'
 |}

Team:LMU-Munich/Notebook/Pathway

From 2010.igem.org

Revision as of 13:40, 29 September 2010

Pathway Notebook

Contents

8-02-2010

8-03-2010

8-04-2010

8-05-2010

8-06-2010

8-07-2010

8-08-2010

8-09-2010

8-10-2010

8-11-2010

8-12-2010

8-13-2010

8-14-2010

8-15-2010

8-16-2010

8-17-2010

8-18-2010

8-19-2010

8-20-2010

8-21-2010

8-22-2010

8-23-2010

8-24-2010

8-25-2010

8-26-2010

8-27-2010

8-28-2010

8-29-2010

8-30-2010

8-31-2010

9-01-2010

9-02-2010

9-03-2010

9-04-2010

9-05-2010

9-06-2010

9-07-2010

9-08-2010

9-09-2010

9-10-2010

9-11-2010

9-12-2010

9-13-2010

9-14-2010

9-15-2010

9-16-2010

9-17-2010

9-18-2010

9-19-2010

9-20-2010

9-21-2010

9-22-2010

9-23-2010

9-24-2010

9-25-2010

9-26-2010

9-27-2010

9-28-2010

9-29-2010

9-30-2010

10-01-2010

10-02-2010

10-03-2010

10-04-2010

10-05-2010

10-06-2010

10-07-2010

10-08-2010

10-09-2010

10-10-2010

10-11-2010

10-12-2010

10-13-2010

10-14-2010

10-15-2010