Team:Stockholm/14 October 2010
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Latest revision as of 11:10, 26 October 2010
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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