Pathway Notebook
Contents
8-02-2010
8-03-2010
Some test text in bold
We created following tests:
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
text
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:
| 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
text
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)
|
| template: 0.45µl (1-6) / 2.5µl (2-5)
|
25µl
| * Extender
|
| MQ: 37.75µl
|
| Puffer10x: 5.4µl
|
| DMSO: 1.25µl
|
| primer forw: 2µl (1-5)
|
| primer rev: 2µl (2-6)
|
| template: 0.45µl (1-6) / 2.5µl (2-5)
|
| dNTP's: 2µl
|
| polymerase: 1µl
|
54µl
PCR-programm:
- 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)
8-27-2010
text
8-28-2010
weekend
8-29-2010
weekend
8-30-2010
text
8-31-2010
text
9-01-2010
text
9-02-2010
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:
- 2: 95°C 30"
- 3: 50°C 30"
- 4: 72°C 2'30"
- 5: repeat step 2-4 35x
|
- 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:
- 2: 95°C 30"
- 3: 50°C 30"
- 4: 72°C 3'
- 5: repeat step 2-4 35x
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:
- 2: 98°C 10"
- 3: Tx 30"
- 4: 72°C 1'
- 5: repeat step 2-4 30x
| 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
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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
weekend
10-03-2010
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