Team:Stockholm/14 October 2010

From 2010.igem.org

(Difference between revisions)
(New page: {{Stockholm/Top2}} ==Andreas== ===Growth curve assay=== New attempt to measure growth curves for CPPs. ====Induction==== *150 ml side-arm E-flasks *15 ml LB *150 μl ON culture (1:100) ...)
 
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Experiment was canceled after 270 min as I was made aware that the spectrophotometer only makes accurate measurements up to &asymp;OD<sub>590</sub> = 0.5.
Experiment was canceled after 270 min as I was made aware that the spectrophotometer only makes accurate measurements up to &asymp;OD<sub>590</sub> = 0.5.
 +
 +
====ON cultures====
 +
New ON cultures set (including two for Mimmi)
 +
*5 ml LB + Amp 100; 37 &deg;C, 225 rpm
 +
**BL21, pEX.SOD&sdot;His
 +
**BL21, pEX.nTra10&sdot;SOD&sdot;His
 +
**BL21, pEX.nTAT&sdot;SOD&sdot;His
 +
**BL21, pEX.nLMWP&sdot;SOD&sdot;His
 +
**(BL21, pEX.SOD)
 +
**(BL21, pEX.yCCS)
 +
 +
==Nina==
 +
 +
===Protein purification===
 +
 +
====Lysis====
 +
 +
I have talked to people in the department that work with protein purifications and they recommended me to start working from a bigger culture such as 40 ml instead of 12 ml.
 +
 +
I used a 40 ml SOD.His (N terminal) culture for this purification.
 +
 +
I performed a batch purification of SOD.His tagg in E.coli BL21. For this I used a 50 ml falcon tube as the column.
 +
 +
*Ni-NTA tube was vortexed to have a homogenized solution of Ni.
 +
*10 ml Ni-NTA was added in the 50 ml falcon tube (column).
 +
*Centrifuged 30 sec at 4000 rpm to remove the supernatant the Ni was dissolved in.
 +
*Added 30 ml lysis buffer without imidazole, DNase and PMSF for equilibration of the Ni-resin. Left on shake in 4 °C for 1 h.
 +
*I prepared the lysis buffer:
 +
 +
For 5 ml pelleted culture use 630 ul lysis buffer
 +
 +
40 ml culture/5 = 8
 +
 +
8 * 630 ul lysis buffer = 5 ml
 +
 +
I double this 5 to 10 ml since I heard at the department that this would be good and there is no harm in using to much of lysis buffer.
 +
 +
Therefore I had 10 ml lysis buffer.
 +
 +
PMSF:
 +
 +
10000 ul/100 = 100 ul (1 mM PMSF) add in lysis buffer
 +
 +
Imidazole:
 +
 +
(10000 ul * 10*10^-3)/2 = 50 ul (10 mM Imidazole) add in lysis buffer
 +
 +
DNase:
 +
 +
10*10^3 ug/ml * Volume = 20 ug/ml * 10 ml 
 +
 +
Volume = 20 ul (20 ug/ml) add in lysis buffer
 +
 +
*Mixed the lysis buffer with the pellet from 40 ml culture & transfered to a glas tube to sonicate for 5 min (75%)
 +
*Transfered the sonicated lysate back to a falcon tube and centrifuged for 20 min 8000 rpm in the cold room.
 +
*performed a ultracentrifugation on the supernatant 40 min 50000 rpm (1 ml of supernatant in each 10 centrifugation tubes with the size of 1 ml). I saved the pellet from both centrifugation steps. The first centrifugation removes the unopen cells and the second centrifugation pellets the membranes and debris from the first centrifugation.
 +
*Centrifuged the column and removed the supernatant that had equilibrated the resin.
 +
*Added the supernatant from the second centrifugation (50000 rpm) to the Ni-resin in the falcon tube. Incubated in 4 °C overnight on shake.
 +
 +
===PCR Colony===
 +
 +
I did a PCR colony screen on the transformations from yesterday.
 +
 +
36 tubes
 +
 +
Master Mix:
 +
 +
*MgCl2 18 ul
 +
*Phusion buffer 5X 180 ul
 +
*dNTP 18 ul
 +
*primerF 54 ul
 +
*primerR 54 ul
 +
*polymerase 18 ul
 +
*water 540 ul
 +
 +
Had 24 ul of the mix in each 36 tubes.
 +
 +
Elongation time for protein A samples: 45 sec and for fusion samples: 2 min.
 +
 +
 +
 +
 +
 +
== Mimmi ==
 +
 +
=== clone non-CPP operon ===
 +
 +
==== Digestion ====
 +
 +
{|
 +
! mix
 +
| pC.S
 +
| pC.Sh
 +
| pC.hS
 +
| pA.RyC
 +
| pMA.Sh
 +
| pMA.hS
 +
|
 +
| pA.RyC
 +
|-
 +
| DNA
 +
| 5.2
 +
| 4.3
 +
| 6.6
 +
| 6.0
 +
| 4.78
 +
| X
 +
| 16.0
 +
|-
 +
| dH<sub>2</sub>O
 +
| 10.8
 +
| 11.7
 +
| 9.4
 +
| 10.0
 +
| 15.22
 +
| 11.85
 +
| X
 +
| 0
 +
|-
 +
| 10xbuffer
 +
| 2
 +
| 2
 +
| 2
 +
| 2
 +
| 2
 +
| 2
 +
| X
 +
| 2
 +
|-
 +
| XbaI
 +
| -
 +
| -
 +
| -
 +
| 1
 +
| -
 +
| -
 +
| X
 +
| 1
 +
|-
 +
| SpeI
 +
| 1
 +
| 1
 +
| 1
 +
| -
 +
| 1.5
 +
| 1.5
 +
| X
 +
| -
 +
|-
 +
| PstI
 +
| 1
 +
| 1
 +
| 1
 +
| 1
 +
| 1
 +
| 1
 +
| X
 +
| 1
 +
|-
 +
| tot
 +
| 20µl
 +
| 20µl
 +
| 20µl
 +
| 20µl
 +
| 20.5µl
 +
| 20.5µl
 +
| X
 +
| 20µl
 +
|}
 +
- c = 50ng/µl
 +
 +
*Incubate in 37&deg;C for 1.5h
 +
 +
 +
==== Ligation ====
 +
 +
{|
 +
! mix
 +
|
 +
|-
 +
| vector
 +
| 1
 +
|-
 +
| insert
 +
| 5
 +
|-
 +
| 10xbuffer
 +
| 2
 +
|-
 +
| H<sub>2</sub>O
 +
| 11
 +
|-
 +
| T4 ligase
 +
| 1
 +
|-
 +
| align="right" | tot
 +
| 20µl
 +
|}
 +
 +
*Incubate in 22&deg;C for 15min
 +
 +
 +
 +
==== Gel extraction ====
 +
(pA.RBS.yCCS    806bp)
 +
 +
*Tube 1.01g
 +
 +
*Tube + gelband 1.11g
 +
 +
 +
 +
==== Transformation ====
 +
 +
* 66µl thawed cells + 2µl ligated vector
 +
 +
*Incubate on ice 30min
 +
*Heat shock 55s in 42&deg;C, cool down on ice
 +
*Add 900µl LB, incubate in 37&deg;C for 45min
 +
*Spinn down cells and remove 900µl supernatant
 +
*Resuspend cells and plate
 +
 +
{{Stockholm/Footer}}

Latest revision as of 11:10, 26 October 2010

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Contents

Andreas

Growth curve assay

New attempt to measure growth curves for CPPs.

Induction

  • 150 ml side-arm E-flasks
  • 15 ml LB
  • 150 μl ON culture (1:100)
    • BL21
    • ON cultures set by Mimmi 13/10
  • 37 °C, 220 rpm

OD590 measurements

  0 min 30 min 60 min 90 min 120 min 150 min 180 min 210 min 240 min 270 min
pEX.SOD⋅His 0.03 0.04 0.10 0.23 0.45 0.78 1.06 1.36 1.56 1.67
pEX.nTra10⋅SOD⋅His 0.03 0.04 0.09 0.19 0.36 0.61 0.94 1.18 1.42 1.56
pEX.nTAT⋅SOD⋅His 0.03 0.05 0.10 0.23 0.49 0.78 1.06 1.35 1.55 1.66
pEX.nLMWP⋅SOD⋅His 0.04 0.05 0.11 0.25 0.52 0.88 1.14 1.40 1.60 1.69

Experiment was canceled after 270 min as I was made aware that the spectrophotometer only makes accurate measurements up to ≈OD590 = 0.5.

ON cultures

New ON cultures set (including two for Mimmi)

  • 5 ml LB + Amp 100; 37 °C, 225 rpm
    • BL21, pEX.SOD⋅His
    • BL21, pEX.nTra10⋅SOD⋅His
    • BL21, pEX.nTAT⋅SOD⋅His
    • BL21, pEX.nLMWP⋅SOD⋅His
    • (BL21, pEX.SOD)
    • (BL21, pEX.yCCS)

Nina

Protein purification

Lysis

I have talked to people in the department that work with protein purifications and they recommended me to start working from a bigger culture such as 40 ml instead of 12 ml.

I used a 40 ml SOD.His (N terminal) culture for this purification.

I performed a batch purification of SOD.His tagg in E.coli BL21. For this I used a 50 ml falcon tube as the column.

  • Ni-NTA tube was vortexed to have a homogenized solution of Ni.
  • 10 ml Ni-NTA was added in the 50 ml falcon tube (column).
  • Centrifuged 30 sec at 4000 rpm to remove the supernatant the Ni was dissolved in.
  • Added 30 ml lysis buffer without imidazole, DNase and PMSF for equilibration of the Ni-resin. Left on shake in 4 °C for 1 h.
  • I prepared the lysis buffer:

For 5 ml pelleted culture use 630 ul lysis buffer

40 ml culture/5 = 8

8 * 630 ul lysis buffer = 5 ml

I double this 5 to 10 ml since I heard at the department that this would be good and there is no harm in using to much of lysis buffer.

Therefore I had 10 ml lysis buffer.

PMSF:

10000 ul/100 = 100 ul (1 mM PMSF) add in lysis buffer

Imidazole:

(10000 ul * 10*10^-3)/2 = 50 ul (10 mM Imidazole) add in lysis buffer

DNase:

10*10^3 ug/ml * Volume = 20 ug/ml * 10 ml

Volume = 20 ul (20 ug/ml) add in lysis buffer

  • Mixed the lysis buffer with the pellet from 40 ml culture & transfered to a glas tube to sonicate for 5 min (75%)
  • Transfered the sonicated lysate back to a falcon tube and centrifuged for 20 min 8000 rpm in the cold room.
  • performed a ultracentrifugation on the supernatant 40 min 50000 rpm (1 ml of supernatant in each 10 centrifugation tubes with the size of 1 ml). I saved the pellet from both centrifugation steps. The first centrifugation removes the unopen cells and the second centrifugation pellets the membranes and debris from the first centrifugation.
  • Centrifuged the column and removed the supernatant that had equilibrated the resin.
  • Added the supernatant from the second centrifugation (50000 rpm) to the Ni-resin in the falcon tube. Incubated in 4 °C overnight on shake.

PCR Colony

I did a PCR colony screen on the transformations from yesterday.

36 tubes

Master Mix:

  • MgCl2 18 ul
  • Phusion buffer 5X 180 ul
  • dNTP 18 ul
  • primerF 54 ul
  • primerR 54 ul
  • polymerase 18 ul
  • water 540 ul

Had 24 ul of the mix in each 36 tubes.

Elongation time for protein A samples: 45 sec and for fusion samples: 2 min.



Mimmi

clone non-CPP operon

Digestion

mix pC.S pC.Sh pC.hS pA.RyC pMA.Sh pMA.hS pA.RyC
DNA 5.2 4.3 6.6 6.0 4.78 X 16.0
dH2O 10.8 11.7 9.4 10.0 15.22 11.85 X 0
10xbuffer 2 2 2 2 2 2 X 2
XbaI - - - 1 - - X 1
SpeI 1 1 1 - 1.5 1.5 X -
PstI 1 1 1 1 1 1 X 1
tot 20µl 20µl 20µl 20µl 20.5µl 20.5µl X 20µl

- c = 50ng/µl

  • Incubate in 37°C for 1.5h


Ligation

mix
vector 1
insert 5
10xbuffer 2
H2O 11
T4 ligase 1
tot 20µl
  • Incubate in 22°C for 15min


Gel extraction

(pA.RBS.yCCS 806bp)

  • Tube 1.01g
  • Tube + gelband 1.11g


Transformation

  • 66µl thawed cells + 2µl ligated vector
  • Incubate on ice 30min
  • Heat shock 55s in 42°C, cool down on ice
  • Add 900µl LB, incubate in 37°C for 45min
  • Spinn down cells and remove 900µl supernatant
  • Resuspend cells and plate





The Faculty of Science at Stockholm University Swedish Vitiligo association (Svenska Vitiligoförbundet) Geneious Fermentas/ Sigma-Aldrich/

Retrieved from "http://2010.igem.org/Team:Stockholm/14_October_2010"