Team:LMU-Munich/Notebook/Pathway

From 2010.igem.org

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joining PCR
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| * hotstar
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| * hotstart
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| MQ: 11.05µl
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| hotstar: 25µl
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| hotstart: 25µl
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| MgCl: 5µl
| MgCl: 5µl

Revision as of 16:11, 13 September 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

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

text

9-07-2010

text

9-08-2010

text

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

text

9-15-2010

text

9-16-2010

text

9-17-2010

text

9-18-2010

weekend

9-19-2010

weekend

9-20-2010

text

9-21-2010

text

9-22-2010

text

9-23-2010

text

9-24-2010

text

9-25-2010

weekend

9-26-2010

weekend

9-27-2010

text

9-28-2010

text

9-29-2010

text

9-30-2010

text

10-01-2010

text

10-02-2010

weekend

10-03-2010

weekend

 

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