Team:Stockholm/14 October 2010
From 2010.igem.org
(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 ≈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 ≈OD<sub>590</sub> = 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 | ||
+ | |- | ||
+ | | 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°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°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 | ||
+ | |||
+ | {{Stockholm/Footer}} |
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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