Team:Stockholm/18 October 2010

From 2010.igem.org

(Difference between revisions)
(Andreas)
(PCR verification for Uppsala-Sweden team)
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PCR run ON.
PCR run ON.
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==Nina==
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===Polyacrylamide gel on SOD===
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I ran a polyacrylamide gel on SOD that I have tried to get pure.
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Ladder: PageRuler Unst. Protein Ladder Fermentas
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[[Image:Mlös1.jpg|200px]]
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Arragement on gel:
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[[Image:Aq6.jpg]]
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I had a Coomassie blue staning and destaining on the gel.
 +
 +
[[Image:Aq5.jpg|250px]]
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 +
===Protein concentration===
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 +
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.
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 +
I got a standard curve from another student (Annika) in the lab I work in.
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 +
[[Image:Aq7.jpg|400px]]

Revision as of 16:23, 24 October 2010


Contents

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

Mlös1.jpg

Arragement on gel:

Aq6.jpg

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

Aq5.jpg

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.

Aq7.jpg