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

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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

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8-11-2010

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8-12-2010

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8-13-2010

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8-14-2010

weekend

8-15-2010

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8-16-2010

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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

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8-21-2010

weekend

8-22-2010

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8-23-2010

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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

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8-27-2010

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8-28-2010

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8-29-2010

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8-30-2010

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8-31-2010

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9-01-2010

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9-02-2010

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9-05-2010

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9-04-2010

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9-05-2010

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9-06-2010

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9-07-2010

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9-08-2010

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9-09-2010

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9-10-2010

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9-11-2010

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9-12-2010

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9-13-2010

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9-14-2010

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9-15-2010

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9-16-2010

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9-17-2010

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9-18-2010

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9-19-2010

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9-20-2010

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9-21-2010

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9-22-2010

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9-23-2010

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9-24-2010

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9-25-2010

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9-26-2010

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9-27-2010

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9-28-2010

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9-29-2010

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9-30-2010

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10-01-2010

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10-02-2010

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10-03-2010

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