Team:Stockholm/1 October 2010

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{{Stockholm/Top2}}
{{Stockholm/Top2}}
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==Andreas==
==Andreas==
===LB agar plates===
===LB agar plates===
*20 x 100 μg/ml Amp LB agar plates
*20 x 100 μg/ml Amp LB agar plates
-
===Assembly of His⋅SOD&sdotcCPP constructs===
+
===Assembly of His⋅SOD⋅cCPP constructs===
Digested pMA.His⋅SOD constructs for later assembly into cCPP plasmids, digested by Johan.
Digested pMA.His⋅SOD constructs for later assembly into cCPP plasmids, digested by Johan.
Line 34: Line 35:
Stored in -20 °C for later ligation.
Stored in -20 °C for later ligation.
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 +
----
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==Nina==
 +
 +
===Miniprep===
 +
 +
I performed a miniprep on IgG protease_Tra10_Ntermin#4 and Fusion_NS#2 according to the procedure that is described in protocols.
 +
 +
===Send for sequencing===
 +
 +
I sent two samples for seqeuncing. The mixture was 15 ul sample and 1.5 ul forward bank vector verification primer VF.
 +
 +
*IgG protease_Tra10_Ntermin#4 ASB0045 682
 +
 +
*Fusion_NS#2 ASB0045 681
 +
 +
===Overnight culture===
 +
 +
I inoculated 12 ml LB and 24 ul chloramphenicol with IgG protease_Tra10_Ntermin#6 again since I acidentally droped the previous sample and therefore lost it.
 +
 +
===iGEM Uppsala collaboration===
 +
 +
I drove to Uppsala and started a collaboration with their team. I obtained a construct that they want me/our group to PCR with verification primers (VF2 & VR) and run on an agarose gel. I in turn gave them the tyrosinase gene to also amplify via PCR but with own designed primers.
 +
 +
*Uppsala iGEM team's PCR master mix and prgm:
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 +
[[Image:Pq.jpg]]
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 +
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== Mimmi ==
 +
 +
=== Over expression ===
 +
 +
*Start culture  (9:30)
 +
**20ml LB + 200µl culture from ON
 +
 +
*At OD=0.6 add IPTG 1mM (checked at 12:00, allready too high OD, had to dilute...)
 +
 +
*Take samples at
 +
**0h
 +
**30min (50min)
 +
**3h
 +
 +
*Spinn down and resuspend in 100µl SDS-buffer
 +
**dilute 3h samples 1:4
 +
**heat at 95°C for 10min
 +
**freeze
 +
 +
*Save pellet from the culture to purify protein
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 +
==Johan==
 +
 +
Realized I had put in the wrong antibiotic in my overnight cultures (doh!). Made new overnight cultures for tomorrow.
 +
 +
{{Stockholm/Footer}}

Latest revision as of 21:07, 27 October 2010

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Contents

Andreas

LB agar plates

  • 20 x 100 μg/ml Amp LB agar plates

Assembly of His⋅SOD⋅cCPP constructs

Digested pMA.His⋅SOD constructs for later assembly into cCPP plasmids, digested by Johan.

Digestion

  pMA.His⋅ SOD
10X FastDigest buffer 3
DNA (3 μg) 7.5
dH2O 17.5
FD EcoRI 1
FD AgeI 1
  30 μl
  • Incubation: 37 °C, 0:30
  • Inactivation: 80 °C, 20 min

Stored in -20 °C for later ligation.

Nina

Miniprep

I performed a miniprep on IgG protease_Tra10_Ntermin#4 and Fusion_NS#2 according to the procedure that is described in protocols.

Send for sequencing

I sent two samples for seqeuncing. The mixture was 15 ul sample and 1.5 ul forward bank vector verification primer VF.

  • IgG protease_Tra10_Ntermin#4 ASB0045 682
  • Fusion_NS#2 ASB0045 681

Overnight culture

I inoculated 12 ml LB and 24 ul chloramphenicol with IgG protease_Tra10_Ntermin#6 again since I acidentally droped the previous sample and therefore lost it.

iGEM Uppsala collaboration

I drove to Uppsala and started a collaboration with their team. I obtained a construct that they want me/our group to PCR with verification primers (VF2 & VR) and run on an agarose gel. I in turn gave them the tyrosinase gene to also amplify via PCR but with own designed primers.

  • Uppsala iGEM team's PCR master mix and prgm:

Pq.jpg




Mimmi

Over expression

  • Start culture (9:30)
    • 20ml LB + 200µl culture from ON
  • At OD=0.6 add IPTG 1mM (checked at 12:00, allready too high OD, had to dilute...)
  • Take samples at
    • 0h
    • 30min (50min)
    • 3h
  • Spinn down and resuspend in 100µl SDS-buffer
    • dilute 3h samples 1:4
    • heat at 95°C for 10min
    • freeze
  • Save pellet from the culture to purify protein

Johan

Realized I had put in the wrong antibiotic in my overnight cultures (doh!). Made new overnight cultures for tomorrow.





The Faculty of Science at Stockholm University Swedish Vitiligo association (Svenska Vitiligoförbundet) Geneious Fermentas/ Sigma-Aldrich/

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