Team:Stockholm/14 October 2010

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(Difference between revisions)
(→ON cultures)
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Elongation time for protein A samples: 45 sec and for fusion samples: 2 min.
Elongation time for protein A samples: 45 sec and for fusion samples: 2 min.
 +
 +
 +
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 +
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== 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
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| 6.6
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| 6.0
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| 4.78
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| X
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| 16.0
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|-
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| dH<sub>2</sub>O
 +
| 10.8
 +
| 11.7
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| 9.4
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| 10.0
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| 15.22
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| 11.85
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| X
 +
| 0
 +
|-
 +
| 10xbuffer
 +
| 2
 +
| 2
 +
| 2
 +
| 2
 +
| 2
 +
| 2
 +
| X
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| 2
 +
|-
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| XbaI
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| -
 +
| -
 +
| -
 +
| 1
 +
| -
 +
| -
 +
| X
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| 1
 +
|-
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| SpeI
 +
| 1
 +
| 1
 +
| 1
 +
| -
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| 1.5
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| 1.5
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| X
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| -
 +
|-
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| PstI
 +
| 1
 +
| 1
 +
| 1
 +
| 1
 +
| 1
 +
| 1
 +
| X
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| 1
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|-
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| tot
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| 20µl
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| 20µl
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| 20µl
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| 20µl
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| 20.5µl
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| 20.5µl
 +
| X
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| 20µl
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|}
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- c = 50ng/µl
 +
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*Incubate in 37&deg;C for 1.5h
 +
 +
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==== Ligation ====
 +
 +
{|
 +
! mix
 +
|
 +
|-
 +
| vector
 +
| 1
 +
|-
 +
| insert
 +
| 5
 +
|-
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| 10xbuffer
 +
| 2
 +
|-
 +
| H<sub>2</sub>O
 +
| 11
 +
|-
 +
| T4 ligase
 +
| 1
 +
|-
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| align="right" | tot
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| 20µl
 +
|}
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 +
*Incubate in 22&deg;C for 15min
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 +
 +
 +
==== Gel extraction ====
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(pA.RBS.yCCS    806bp)
 +
 +
*Tube 1.01g
 +
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*Tube + gelband 1.11g
 +
 +
 +
 +
==== Transformation ====
 +
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* 66µl thawed cells + 2µl ligated vector
 +
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*Incubate on ice 30min
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*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

Revision as of 23:38, 25 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
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