Pathway Notebook

Contents

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

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

-> no products on gel picture

8-10-2010

PCR were performed with pdu-template:

mastermix:

template: 0.5µl

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

template: 2µl

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:

-> 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

25µl

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

template: 10µl

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

template: 2µl / 4µl

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

template: 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

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

template: 10µl

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'

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

• 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'

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

text

9-18-2010

weekend

9-19-2010

weekend

9-20-2010

text

9-21-2010

text

9-22-2010

text

9-23-2010

text

9-24-2010

text

9-25-2010

weekend

9-26-2010

weekend

9-27-2010

text

9-28-2010

text

9-29-2010

text

9-30-2010

text

10-01-2010

text

10-02-2010

weekend

10-03-2010

weekend