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Revision as of 15:53, 15 October 2010 (view source) AndreasConstantinou (Talk | contribs) (→Andreas) ← Older edit		Revision as of 12:13, 25 October 2010 (view source) NinaSchiller (Talk | contribs) (→Andreas) Newer edit →
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	{{Stockholm/Top2}}		{{Stockholm/Top2}}
		+	==Nina==
		+
		+	===Continuation of protein purification===
		+
		+	====Washing====
		+
		+	I washed the column with 10 X volumes of wash buffer.
		+
		+	The Ni-resin (in the form of pellet) is 10 ml, therefore 10 * 10ml Ni-NTA = 100 ml wash buffer.
		+
		+	The imidazole is 2 M.
		+
		+	2M * Volume = 100 * 30 *10^-3 M  Volume imidaziole that needs to be added in the wash 100 ml is 1.5 ml
		+
		+	Washing:
		+
		+	[[Image:Aq14.jpg|300px]]
		+
		+	====Elution====
		+
		+	I eluted the column with 5 X volumes of elution buffer.
		+
		+	The Ni-resin (in the form of pellet) is 10 ml, therefore 5 * 10ml Ni-NTA = 50 ml elution buffer.
		+
		+	*20 ml 40 min on shake 4°C
		+	*10 ml 30 min on shake 4°C
		+	*10 ml 30 min on shake 4°C
		+	*10 ml 30 min on shake 4°C
		+
		+	I saved these samples in the cold room 4°C for tomorrow.
		+
		+	===Agarose gel verification===
		+
		+	I ran the PCR products on an 1 % agarose gel 80 V to check if I had any inserts.
		+
		+	Ladder: MassRuler™ DNA Ladder Mix, ready-to-use, 80-10,000 bp
		+
		+	[[Image:Aq12.jpg|150px]]
		+
		+	Arrangement on gel:
		+
		+	[[Image:Aq13.jpg]]
		+
		+	Gels:
		+
		+	[[Image:Aq15.jpg|250px]]
		+
		+	Unfortunately non of the bands show that there has been an insert, it might be that the transformation into BL21 cells was not a good idea since they are not cloning cells, but we are short on time and I tried to make a short cut. It might have failed this time. I will run a new screen with 16 colonies/dish, it that won't work then this cloning has not worked out well.
		+
	==Andreas==		==Andreas==
	===Growth curve assay===		===Growth curve assay===

---

Revision as of 12:13, 25 October 2010

Contents 1 Nina 1.1 Continuation of protein purification 1.1.1 Washing 1.1.2 Elution 1.2 Agarose gel verification 2 Andreas 2.1 Growth curve assay 2.1.1 Setup 2.1.2 OD590 measurements 2.1.3 Results

Nina

Continuation of protein purification

Washing

I washed the column with 10 X volumes of wash buffer.

The Ni-resin (in the form of pellet) is 10 ml, therefore 10 * 10ml Ni-NTA = 100 ml wash buffer.

The imidazole is 2 M.

2M * Volume = 100 * 30 *10^-3 M Volume imidaziole that needs to be added in the wash 100 ml is 1.5 ml

Washing:

Elution

I eluted the column with 5 X volumes of elution buffer.

The Ni-resin (in the form of pellet) is 10 ml, therefore 5 * 10ml Ni-NTA = 50 ml elution buffer.

• 20 ml 40 min on shake 4°C
• 10 ml 30 min on shake 4°C
• 10 ml 30 min on shake 4°C
• 10 ml 30 min on shake 4°C

I saved these samples in the cold room 4°C for tomorrow.

Agarose gel verification

I ran the PCR products on an 1 % agarose gel 80 V to check if I had any inserts.

Ladder: MassRuler™ DNA Ladder Mix, ready-to-use, 80-10,000 bp

Arrangement on gel:

Gels:

Unfortunately non of the bands show that there has been an insert, it might be that the transformation into BL21 cells was not a good idea since they are not cloning cells, but we are short on time and I tried to make a short cut. It might have failed this time. I will run a new screen with 16 colonies/dish, it that won't work then this cloning has not worked out well.

Andreas

Growth curve assay

Setup

Inoculated new flasks from ON cultures set 14/10:

• 250 ml E-flasks
• 25 ml LB + Amp 100 + 1 mM IPTG
• 250 μl ON culture
  1. BL21 pEX.SOD⋅His
  2. BL21 pEX.nTra10⋅SOD⋅His
  3. BL21 pEX.nTAT⋅SOD⋅His
  4. BL21 pEX.nLMWP⋅SOD⋅His
• 37 °C, 225 rpm

OD₅₉₀ measurements made every 30 min with appropriate dilution in fresh LB.

OD₅₉₀ measurements

	0 min	30 min	60 min	90 min	120 min	150 min	180 min	210 min	240 min	270 min	300 min	330 min	360 min	390 min
pEX.SOD⋅His	0.035	0.047	0.090	0.180	0.195	0.176	0.251	0.177	0.239	0.140	0.164	0.205	0.196	0.193
pEX.nTra10⋅SOD⋅His	0.037	0.050	0.092	0.163	0.156	0.132	0.175	0.130	0.164	0.116	0.155	0.167	0.154	0.168
pEX.nTAT⋅SOD⋅His	0.033	0.046	0.090	0.170	0.177	0.161	0.225	0.153	0.225	0.045	0.170	0.162	0.193	0.200
pEX.nLMWP⋅SOD⋅His	0.031	0.042	0.86	0.174	0.192	0.178	0.237	0.170	0.242	0.137	0.180	0.185	0.200	0.200
Dilution factor	1	1	1	1	2	4	5	10	10	20	20	20	20	20

Results

Results indicate no significant difference in growth between different cultures. Slightly lower curve for the nTra10-expressing clone, which is somewhat interesting, since a similar progress was seen in the canceled experiment from 14/10. At least one more experiment will be run next week, using new BL21 clones; this will show whether this is just a coincidence or not.