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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

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)

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

* hotstar

MQ: 11.05µl

hotstar: 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

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

1&3	2&4
PCRmastermix: 10µl	10µl
primer rev: 1.5µl	1.5µl
primer fwd: 1.5µl	1.5µl
template: 2µl	4µl
DMSO: 0.5µl	0.5µl
MQ: 4.5µl	2.5µl
sum: 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'

From 2010.igem.org

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