Team:Stockholm/14 October 2010

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Revision as of 12:55, 25 October 2010

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.

@@ Line 87: / Line 87: @@
 **(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
+ml culture/5 = 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:
+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:
+*10^3 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.
+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.