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
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:
| 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)
|
| 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
| 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:
- 2: 94°C 30"
- 3: 52°C 30"
- 4: 73°C 3'
- 5: repeat step 2-4 35x
|
|
=> no fragments
| 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:
- 2: 95°C 30"
- 3: 52°C 30"
- 4: 72°C 2'30"
- 5: repeat step 2-4 30x
|
|
=> no fragments with right size
| 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:
- 2: 95°C 30"
- 3: 50°C 30"
- 4: 72°C 2'30"
- 5: repeat step 2-4 35x
|
|
- 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:
- 2: 95°C 30"
- 3: 54°C 30"
- 4: 72°C 2'30"
- 5: repeat step 2-4 35x
|
|
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
|
- 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:
- 2: 98°C 30"
- 3: 50°C 30"
- 4: 72°C 2'30"
- 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
| 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
| 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
|
| 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
| 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
| 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
|
| 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 amplification of pdu-operon
with PCRmastermix
| PCRmastermix
| 10µl
|
| primer fwd
| 1.5µl
|
| primer rev
| 1.5µl
|
| template (Knutplasmid)
| 2µl
|
| DMSO
| 0.5µl
|
| MQ
| 4.5µl
|
| sum
| 20µl
|
PCRprogramm
94°C 2'
|
| 94°C 30"
|
| 52°C 30"
|
| 72°C 5'
|
repeat 30x
|
| 72°C 10'
|
test amplification of pdu-operon
with PCRextender
5 charges:
| buffer
| 2µl
|
| primer fwd
| 1µl
|
| primer rev
| 1µl
|
| template
| 2µl
|
| dNTP's
| 2µl
|
| DMSO
| 0.5µl
|
| MQ
| 11µl
|
| PCRextender pol
| 0.5µl
|
| sum
| 20µl
|
| charge
| 1
| 2
| 3
| 4
| 5
|
| Tx[°C]
| 50
| 51.4
| 54.8
| 60.2
| 63.6
|
PCRprogramm
94°C 2'
|
| 94°C 20"
|
| Tx 20"
|
| 72°C 3'
|
repeat 30x
|
| 72°C 10'
|
colony PCR
for BioBricks I 712074 and E 0240 with PCRmastermix
| primer fwd
| 6µl
|
| primer rev
| 6µl
|
| DMSO
| 2µl
|
| MQ
| 26µl
|
| PCRmastermix
| 40µl
|
| sum
| 80µl
|
PCRprogramm
94°C 2'
|
| 94°C 30"
|
| 52°C 30"
|
| 72°C 3'
|
repeat 30x
|
| 72°C 10'
|
GELPHOTO REINSTELLEN
9-22-2010
1. testamplification of pdu-operon (fragment 1/2)
with PCRextender
| buffer
| 5µl
|
| primer#1
| 2µl
|
| primer#6
| 2µl
|
| template (fragment1/2)
| 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'
|
2. testamplification of pdu-operon (fragment 3/4)
with PCRextender
| buffer
| 5µl
|
| primer#2
| 2µl
|
| primer#5
| 2µl
|
| template
| 20µl
|
| dNTP's
| 3µl
|
| DMSO
| 1.5µl
|
| MQ
| 16µl
|
| PCRextender polymerase
| 0.5µl
|
| sum
| 50µl
|
PCRprogramm
94°C 2'
|
| 94°C 20"
|
| 52°C 20"
|
| 72°C 5'
|
repeat 30x
|
| 72°C 10'
|
3. digest of BoBricks
|
| enzym1
| enzym2
| buffer
|
| I 712074
| EcoRI
| SpeI
| MC
|
| E 0240
| XbaI
| PstI
| D+MC
|
| ccdB Kan
| EcoRI
| PstI
| H
|
| buffer
| 2µl
|
| enzym1
| 0.5µl
|
| enzym2
| 0.5µl
|
| BSA
| 0.5µl
|
| DNA
| 17µl
|
| sum
| 20.5µl
|
=> no right fragments visible
9-23-2010
digest of BioBricks
with OpenWetware-protocoll " Knight: restriction digest"
|
| Enzym1
| Enzym2
| buffer
| quantity of DNA(x)
| antibiotic resistance
|
| I 712074
| EcoRI
| SpeI
| MC
| 40µl
| Amp
|
| E 0420
| XbaI
| PstI
| D
| 40µl
| Kam (A/K)
|
| ccdB tet
| EcoRI
| PstI
| H
| 10µl
| Tet
|
| buffer
| 5µl
|
| BSA
| 1µl
|
| enzym1/2
| each 1µl
|
| DNA
| x (look table)
|
| MQ
| 42-xµl
|
| sum
| 50µl
|
joiningPCR - pdu-operon (fragment3/4)
with PCRextender
| buffer
| 5µl
|
| primer fwd
| 2µl
|
| primer rev
| 2µl
|
| fragment4
| 5µl
|
| fragment3
| 4.5µl
|
| dNTP's
| 3µl
|
| DMSO
| 1.5µl
|
| MQ
| 26.5µl
|
| PCRextender polymerase
| 0.5µl
|
| sum
| 50µl
|
PCRprogramm
94°C 2'
|
| 94°C 20"
|
| 52°C 20"
|
| 72°C 5'
|
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'
|
1. amplification of lctO
| primer fwd
| 1.5µl
|
| primer rev
| 1.5µl
|
| template (gDNA lactococcus lactus)
| 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'
|
2. amplification of aurF
-
| 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'
|
3. amplification of pduD
with PCRmastermix
| primer fwd
| 1.5µl
|
| primer rev
| 1.5µl
|
| template (gDNA citrobacter freundii)
| 2µl
|
| DMSO
| 0.5µl
|
| MQ
| 4.5µl
|
| PCRmastermix
| 10µl
|
| sum
| 20µl
|
=> 2 charges each 10µl
PCRprogramm
94°C 2'
|
| 94°C 30"
|
| 52°C 30"
|
| 72°C 2'
|
repeat 30x
|
| 72°C 5'
|
4. amplification of pduC
with PCRmastermix
| primer fwd
| 1.5µl
|
| primer rev
| 1.5µl
|
| template (gDNA citrobacter freundii)
| 2µl
|
| DMSO
| 0.5µl
|
| MQ
| 4.5µl
|
| PCRmastermix
| 10µl
|
| sum
| 20µl
|
=> 2 charges each 10µl
PCRprogramm
94°C 2'
|
| 94°C 30"
|
| 52°C 30"
|
| 72°C 3'
|
repeat 30x
|
| 72°C 5'
|
GELPHOTO REINSTELLEN
9-24-2010
amplification of pduD
with PCRmastermix
| template
| 1µl
|
| MasterMix
| 5µl
|
| Primer *2
| 0.75µl *2
|
| DMSO
| 0.25µl
|
| H2O
| 2.25µl
|
| sum
| 10µl
|
PCRprogramm:
94°C 2'
|
| 94°C 30"
|
| 52°C 30"
|
| 72°C 2'
|
repeat 30x
|
| 72°C 5'
|
amplification of pduC
same charge as before(pduD)
PCRprogramm:
94°C 2'
|
| 94°C 30"
|
| 52°C 30"
|
| 72°C 3'
|
repeat 30x
|
| 72°C 5'
|
ligation of the BioBricks
|
| T4buffer (10x)
| Vector(30ng/µl)
| Insert1(20ng/µl)
| Insert2(15ng/µl)
| MQ
| T4 Ligase
| sum
|
| Charge1
| 2µl
| 2µl
| 6µl
| 8µl
| 1µl
| 1µl
| 20µl
|
| Charge2
| 2µl
| 1µl
| 6µl
| 8µl
| 1µl
| 2µl
| 20µl
|
- 30min at roomtemperature
- 10min denaturation at 65°C
joiningPCR - pduOperon fragment(3/4)
with PCRextender
| buffer
| 5µl
|
| primer fwd
| 2µl
|
| primer rev
| 2µl
|
| fragment4
| 4.5µl(20ng/µl)
|
| fragment3
| 5µl(15ng/µl)
|
| dNTP's
| 3µl
|
| DMSO
| 1.5µl
|
| MQ
| 26.5µl
|
| PCRextender polymerase
| 0.5µl
|
| sum
| 50µl
|
charge 3-5 s
PCRprogramm:
without primers
94°C 2'
|
| 94°C 20"
|
| 52°C 20"
|
| 72°C 5'
|
repeat 10x
|
| 72°C 10'
|
edit primers
94°C 2'
|
| 94°C 20"
|
| 52°C 20"
|
| 72°C 5'
|
repeat 30x
|
| 72°C 10'
|
joiningPCR - pduOperon fragment(3/4)
same as before, but editing primers after 5 cyles
amplification of pduD
PCR with gradient
|
| PCRmastermix
| template
| MQ
| primer fwd
| primer rev
| DMSO
| sum
|
| D2
| 8x10µl
| 8x2µl
| 8x4.5µl
| 8x1.5µl
| 8x1.5µl
| 8x0.5µl
| each20µl
|
| D4
| 8x10µl
| 8x4µl
| 8x2.5µl
| 8x1.5µl
| 8x1.5µl
| 8x0.5µl
| each20µl
|
PCRprogramm:
94°C 2'
|
| 94°C 30"
|
| Tx°C 30"
|
| 72°C 1'40"
|
repeat 30x
|
| 72°C 5'
|
| charge
| D2.1
| D2.2
| D2.3
| D2.4
| D2.5
| D2.6
| D2.7
| D2.8
| D4.1
| D4.2
| D4.3
| D4.4
| D4.5
| D4.6
| D4.7
| D4.8
|
| Tx
| 45
| 47.2
| 52.4
| 55.2
| 57.8
| 60.6
| 65.8
| 68
| 45
| 47.2
| 52.4
| 55.2
| 57.8
| 60.6
| 65.8
| 68
|
GELPHOTO REINSTELLEN
9-25-2010
weekend
9-26-2010
weekend
9-27-2010
transformation of KNUT(pduOperon)into BL21
9-28-2010
9-29-2010
text
9-30-2010
text
10-01-2010
text
10-02-2010
weekend
10-03-2010
weekend
10-04-2010
text
10-05-2010
text
10-06-2010
text
10-07-2010
text
10-08-2010
text
10-09-2010
weekend
10-10-2010
weekend
10-11-2010
text
10-12-2010
text
10-13-2010
text
10-14-2010
text
10-15-2010
text
10-16-2010
weekend
10-17-2010
weekend
10-18-2010
text
10-19-2010
text
10-20-2010
text
10-21-2010
text
10-22-2010
text
10-23-2010
weekend
10-24-2010
weekend
10-25-2010
text
10-26-2010
text
10-27-2010
text
10-28-2010
text
10-29-2010
text
10-30-2010
weekend
10-31-2010
weekend
|
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