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Revision as of 18:21, 18 October 2010 (view source) AndreasConstantinou (Talk | contribs) (→Andreas) ← Older edit		Revision as of 16:23, 24 October 2010 (view source) NinaSchiller (Talk | contribs) (→PCR verification for Uppsala-Sweden team) Newer edit →
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	PCR run ON.		PCR run ON.
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		+	==Nina==
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		+	===Polyacrylamide gel on SOD===
		+
		+	I ran a polyacrylamide gel on SOD that I have tried to get pure.
		+
		+	Ladder: PageRuler Unst. Protein Ladder Fermentas
		+
		+	[[Image:Mlös1.jpg|200px]]
		+
		+	Arragement on gel:
		+
		+	[[Image:Aq6.jpg]]
		+
		+	I had a Coomassie blue staning and destaining on the gel.
		+
		+	[[Image:Aq5.jpg|250px]]
		+
		+	===Protein concentration===
		+
		+	I measured the protein conc on the samples: unbound, wash 1-3, elute 1-4 and not pure to check if I had any proteins. I had to test this since the gel shows that I don't have any proteins, even not much in the unbound lane, which should have a lot of bands. It could also be that my samples that I loaded onto the gel was very diluted since I had fractions consisting of 10 ml solution. This could give me an indication wheter I need to concentrate my fractions with a Millipore centrifugation.
		+
		+	I measured the concentrations using a BSA-method.
		+
		+	I got a standard curve from another student (Annika) in the lab I work in.
		+
		+	[[Image:Aq7.jpg|400px]]

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Revision as of 16:23, 24 October 2010

Contents 1 Andreas 1.1 Transfer of ProtA⋅His to pEX 1.1.1 Digestion 1.1.2 Ligation 1.1.3 Transformation 1.2 PCR verification for Uppsala-Sweden team 2 Nina 2.1 Polyacrylamide gel on SOD 2.2 Protein concentration

Andreas

Transfer of ProtA⋅His to pEX

Digestion

	Sample
10X FastDigest buffer	1
Plasmid DNA	7
dH2O	0
FD XbaI	1
FD PstI	1
	10 μl

• Incubation: 37 °C, 0:30
• Inactivation: 80 °C, 3:00

De-phosphorylated pre-digested/extracted pEX vector:

• 8 μl extracted and digested (X+P) pEX vector DNA
• 1 μl FD buffer
• 1 μl FastAP (alkaline phosphatase)

• Incubation: 37 °C, 0:30
• Inactivation: 80 °C, 3:00

Ligation

10X T4 Ligase buffer	2
Vector DNA	1
Insert DNA	11
dH2O	5
T4 DNA ligase	1
	20 μl

• Incubation: 22 °C, 1 h

Transformation

Including three more transformations

Modified quick-transformation protocol:

• 30 min on ice
• 50 μl BL21
  1. 2 μl pEX.ProtA⋅his ligation mix
  2. 0.5 μl pEX.IgGp
  3. 0.5 μl pEX.SOD.RyC
  4. 0.5 μl pEX.hS.RyC

PCR verification for Uppsala-Sweden team

Helping the Uppsala team with a PCR verification of one of their assemblies.

Summed up their total construct length in pSB1x3 to 6566 bp.

• K1: C2 & C4 (x2)
• K2: C2 & C5 (x2)

Standard colony PCR settings:

• Elongation time: 10 min
• Annealing temp: 55 °C and 60 °C

PCR run ON.

Nina

Polyacrylamide gel on SOD

I ran a polyacrylamide gel on SOD that I have tried to get pure.

Ladder: PageRuler Unst. Protein Ladder Fermentas

Arragement on gel:

I had a Coomassie blue staning and destaining on the gel.

Protein concentration

I measured the protein conc on the samples: unbound, wash 1-3, elute 1-4 and not pure to check if I had any proteins. I had to test this since the gel shows that I don't have any proteins, even not much in the unbound lane, which should have a lot of bands. It could also be that my samples that I loaded onto the gel was very diluted since I had fractions consisting of 10 ml solution. This could give me an indication wheter I need to concentrate my fractions with a Millipore centrifugation.

I measured the concentrations using a BSA-method.

I got a standard curve from another student (Annika) in the lab I work in.