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	SOD.his.RBS.yCCS 2		SOD.his.RBS.yCCS 2
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Latest revision as of 11:10, 26 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 3 Mimmi 3.1 clone non-CPP operon 3.1.1 colony PCR 3.1.2 Gel 3.1.3 ON culture

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.

Mimmi

clone non-CPP operon

colony PCR

mix		x16		primers		samples
mastermix	24.5			pSB_VF2		pSB1C3.SOD.RBS.yCCS 1-5
DNA	0.5			pSB_VR		pSB1C3.SOD.his.RBS.yCCS 1-5
tot	25µl					pSB1C3.his.SOD.RBS.yCCS 1-5
						positive control (pSB1C3.LMWP.SOD.his)

Gel

well	sample	well	sample
1	ladder	12	ladder
2	pC.S_yC 1	13	pC.hS_yC 1
3	pC.S_yC 2	14	pC.hS_yC 2
4	pC.S_yC 3	15	pC.hS_yC 3
5	pC.S_yC 4	16	pC.hS_yC 4
6	pC.S_yC 5	17	pC.hS_yC 5
7	pC.Sh_yC 1	18	pC.L.hS, control
8	pC.Sh_yC 2
9	pC.Sh_yC 3
10	pC.Sh_yC 4
11	pC.Sh_yC 5

ON culture

3ml LB Cm₂₅ + toothpick from colony

SOD.RBS.yCCS 5 SOD.his.RBS.yCCS 2 his.SOD.RBA.yCCS 3