Team:LMU-Munich/Notebook/Pathway

From 2010.igem.org

(Difference between revisions)
(→9-21-2010)
(→10-04-2010)
 
(14 intermediate revisions not shown)
Line 1,729: Line 1,729:
== 9-21-2010 ==
== 9-21-2010 ==
 +
<font color="#009933">test amplification of pdu-operon</font>
 +
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'
 +
|}
 +
 +
<font color="#009933">test amplification of pdu-operon</font>
 +
 +
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
 +
|-
 +
|}
 +
 +
{| class="wikitable" border="1"
 +
|-
 +
! 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'
 +
|}
 +
 +
<font color="#009933">colony PCR</font>
 +
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 ==
 +
 +
<font color="#009933">1. testamplification of pdu-operon</font> (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'
 +
|}
 +
 +
 +
<font color="#009933">2. testamplification of pdu-operon</font> (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'
 +
|}
 +
 +
<font color="#009933">3. digest of BoBricks</font>
 +
 +
{| class="wikitable" border="1"
 +
|-
 +
!
 +
! 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 ==
 +
 +
<font color="#009933">digest of BioBricks</font>
 +
 +
with OpenWetware-protocoll " Knight: restriction digest"
 +
 +
{| class="wikitable" border="1"
 +
|-
 +
!
 +
! 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
 +
|}
 +
 +
<font color="#009933">joiningPCR - pdu-operon</font> (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'
 +
|}
<font color="#009933">test PCRamplification of pdu-operon</font> (fragment1/2)
<font color="#009933">test PCRamplification of pdu-operon</font> (fragment1/2)
with PCRextender
with PCRextender
Line 1,977: Line 2,387:
GELPHOTO REINSTELLEN
GELPHOTO REINSTELLEN
-
== 9-22-2010 ==
 
-
text
 
-
== 9-23-2010 ==
 
-
text
 
== 9-24-2010 ==
== 9-24-2010 ==
-
text
+
 
 +
<font color="#009933">amplification of pduD</font>
 +
with PCRmastermix
 +
::{|
 +
|-
 +
|template
 +
|1µl
 +
|-
 +
|MasterMix
 +
|5µl
 +
|-
 +
|Primer *2
 +
|0.75µl *2
 +
|-
 +
|DMSO
 +
|0.25µl
 +
|-
 +
|H<sub>2</sub>O
 +
|2.25µl
 +
|-
 +
|sum
 +
|10µl
 +
|-
 +
|}
 +
 
 +
PCRprogramm:
 +
::{|
 +
|94°C 2'
 +
----
 +
|-
 +
|94°C 30"
 +
|-
 +
|52°C 30"
 +
|-
 +
|72°C 2'
 +
|-
 +
|repeat 30x
 +
----
 +
|-
 +
|72°C 5'
 +
|}
 +
 
 +
<font color="#009933">amplification of pduC</font>
 +
 
 +
same charge as before(pduD)
 +
 
 +
PCRprogramm:
 +
::{|
 +
|94°C 2'
 +
----
 +
|-
 +
|94°C 30"
 +
|-
 +
|52°C 30"
 +
|-
 +
|72°C 3'
 +
|-
 +
|repeat 30x
 +
----
 +
|-
 +
|72°C 5'
 +
|}
 +
 
 +
 
 +
 
 +
<font color="#009933">ligation of the BioBricks</font>
 +
 
 +
{| class="wikitable" border="1"
 +
|-
 +
!
 +
! 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
 +
 
 +
 
 +
<font color="#009933">joiningPCR - pduOperon</font> 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'
 +
|}
 +
 
 +
<font color="#009933">joiningPCR - pduOperon</font> fragment(3/4)
 +
 
 +
same as before, but editing primers after 5 cyles
 +
 
 +
 
 +
<font color="#009933">amplification of pduD</font>
 +
PCR with gradient
 +
{| class="wikitable" border="1"
 +
|-
 +
!
 +
! 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'
 +
|}
 +
 
 +
{| class="wikitable" border="1"
 +
|-
 +
! 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 ==
== 9-25-2010 ==
weekend
weekend
Line 1,989: Line 2,661:
== 9-27-2010 ==
== 9-27-2010 ==
-
text
+
transformation of KNUT(pduOperon)into BL21
 +
 
== 9-28-2010 ==
== 9-28-2010 ==
-
text
+
 
== 9-29-2010 ==
== 9-29-2010 ==
text
text
Line 1,997: Line 2,670:
text
text
== 10-01-2010 ==
== 10-01-2010 ==
-
text
+
<font color="#009933">in vivo verification of AurF</font>
 +
 
 +
''streptomyces thioluteus'' culture in LB
 +
4-aminosalicylacid-gradient and negativ controll:
 +
{| class="wikitable" border="1"
 +
|-
 +
! PABA
 +
! 0.5%
 +
! 1%
 +
! 2.5%
 +
! 5%
 +
! 0%
 +
|}
 +
 
 +
1ml cultures; 0.5%=>5mg 5%=>50mg
 +
 
 +
after 2h, 37°C:
 +
* 0%, 0.5%, 1% show great growth
 +
* 2.5%, 5% inhibition of growth
 +
 
 +
=> more 4-aminosalicylacid comes to more orange product, but also minus cellquantity
 +
 
== 10-02-2010 ==
== 10-02-2010 ==
weekend
weekend
Line 2,005: Line 2,699:
== 10-04-2010 ==
== 10-04-2010 ==
-
text
+
same as on friday, but charges 25ml SOB, 50µl MgCl<sub>2</sub> solution (0.2g/ml)
 +
 
== 10-05-2010 ==
== 10-05-2010 ==
text
text

Latest revision as of 15:26, 4 October 2010


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
40 10-04-2010 10-05-2010 10-06-2010 10-07-2010 10-08-2010 10-09-2010 10-10-2010
41 10-11-2010 10-12-2010 10-13-2010 10-14-2010 10-15-2010 10-16-2010 10-17-2010
42 10-18-2010 10-19-2010 10-20-2010 10-21-2010 10-22-2010 10-23-2010 10-24-2010
43 10-25-2010 10-26-2010 10-27-2010 10-28-2010 10-29-2010 10-30-2010 10-31-2010

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

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 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

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:

  • 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'
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

  • 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

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

  • with PCRextender


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

  • with PCRmastermix


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

in vivo verification of AurF

streptomyces thioluteus culture in LB 4-aminosalicylacid-gradient and negativ controll:

PABA 0.5% 1% 2.5% 5% 0%

1ml cultures; 0.5%=>5mg 5%=>50mg

after 2h, 37°C:

  • 0%, 0.5%, 1% show great growth
  • 2.5%, 5% inhibition of growth

=> more 4-aminosalicylacid comes to more orange product, but also minus cellquantity

10-02-2010

weekend

10-03-2010

weekend


10-04-2010

same as on friday, but charges 25ml SOB, 50µl MgCl2 solution (0.2g/ml)

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

 

Retrieved from "http://2010.igem.org/Team:LMU-Munich/Notebook/Pathway"