Team:LMU-Munich/Notebook/Pathway
From 2010.igem.org
(→9-09-2010) |
(→9-09-2010) |
||
Line 891: | Line 891: | ||
| 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 | ||
+ | |- | ||
+ | |charge | ||
+ | |20µl | ||
+ | |20µl | ||
+ | |20µl | ||
+ | |20µl | ||
|}::{| | |}::{| | ||
|1&3 | |1&3 |
Revision as of 11:30, 10 September 2010
Some test text in bold
We created following tests:
Example of a table
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
text
weekend
weekend
PCR were performed as follows:
mastermix:
-> no products on gel picture
PCR were performed with pdu-template:
mastermix:
this mix was produced for all of the six primer pairs (see 9.8.10)
text
text
text
weekend
weekend
text
PCR mastermix
text
PCR:
-> each 25µl
transformation efficiency from competent cells: 5.6x106
PCR mastermix
>-
weekend
weekend
text
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
- done with protocoll (3 Transformation)
-> we tested with the following DNA:
-> as the cells are probably in a to high concentration, we thined them down with saline to an end-concentration of 10 -2
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
- done with protocoll (3 Transformation)
-> we tested with the following DNA: pUC (10pn/µl, Amp) 1 µl was used
joining PCR
25µl
54µl
PCR-programm:
text
weekend
weekend
text
=> 50µl
PCR-programm:
=> no fragments
=> 50µl
PCR-programm:
=> no fragments with right size
=> no fragments
primer 5+8
=> testing the template; 2 equal charges to check template
each charge 20µl
PCR-programm:
=> 20µl
PCR-programm:
primer2+5
each charge 20µl
PCR-programm:
each charge 20µl
primer combinations:
PCR-programm:
100µl => each charge 25µl
primer combinations:
PCR-programm:
PCR- amplification of pdu
primer 5+8
100µl => 5 charges, each 20µl
PCR-programm with temperature gradient:
weekend
weekend
text
text
text
PCR amplification of pduD from Citrobacter freundii
PCR programm for charge 1&2:
PCR programm for charge 3&4:
PCRamplification of pduD
|1&3
|2&4
|-
| 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
|}
PCR programm
PCR amplification of pduD from Citrobacter freundii
PCR programm:
weekend
weekend
text
text
text
text
text
weekend
weekend
text
text
text
text
text
weekend
weekend
text
text
text
text
text
weekend
weekend
Pathway Notebook
Contents
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
8-03-2010
8-04-2010
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
8-06-2010
8-07-2010
8-08-2010
8-09-2010
MQ: 93.6µl
10xbuffer: 12.0µl
dNTP's: 2.4µl (each 10mM)
Phusion: 1.2µl (Pfu-Promega)
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
8-10-2010
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
8-11-2010
8-12-2010
8-13-2010
8-14-2010
8-15-2010
8-16-2010
8-17-2010
MQ: 34.4µl
10xbuffer: 5µl
pF: 3µl
pR: 3µl
template: 2µl
dNTP's: 2µl
Pfu(GeneON): 0.6µl
8-18-2010
8-19-2010
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
8-20-2010
MQ: 24.75µl
DMSO: 1.25µl
10xbuffer: 5µl
pF: 3µl
pR: 3µl
template: 10µl
dNTP's: 2µl
Pfu(GeneON): 1µl
8-21-2010
8-22-2010
8-23-2010
8-24-2010
Test-Transformation
in order to see whether the cells will work
8-25-2010
Test-Transformation
in order to see whether the cells will work
8-26-2010
* 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)
* 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
8-27-2010
8-28-2010
8-29-2010
8-30-2010
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
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
primer combinations: 1+6 & 2+5
in the same way as PCR1&2
9-01-2010
PCRmastermix(2x): 10µl
primer fwd: 1.5µl
primer rev: 1.5µl
template: 2µl / 4µl
DMSO: 0.5µl
MQ: 4.5µl / 2.5µl
PCRmastermix(2x): 10µl
primer fwd: 1.5µl
primer rev: 1.5µl
template: 5µl
DMSO: 0.5µl
MQ: 1.5µl
9-02-2010
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
PCRmastermix: 10µl (Taq-polymerase)
primer fwd: 1.5µl
primer rev: 1.5µl
template[ng/µl]: 4µl
MQ: 2.5µl
DMSO: 0.5µl
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
9-03-2010
MQ: 51.5µl
buffer5x: 20µl
primer fwd: 5µl
primer rev: 5µl
dNTP's: 5µl
DMSO: 2.5µl
template: 10µl
Phusion polymerase: 1µl
charge
1
2
3
4
5
Tx [°C]
50
52.5
56.4
61
64.7
9-04-2010
9-05-2010
9-06-2010
9-07-2010
9-08-2010
9-09-2010
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
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'
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'
::{|
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
charge
20µl
20µl
20µl
20µl
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
PCRmastermix: 10µl (+Taq)
primer fwd: 1.5µl
primer rev: 1.5µl
template: 2µl
DMSO: 0.5µl
MQ: 4.5µl
sum
20µl
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'
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