Team:Stockholm/14 August 2010

From 2010.igem.org

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(Transform the ligate samples)
 
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==Nina==
==Nina==
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==Mini prep on Tyrosinase==
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===Mini prep on Tyrosinase===
I performed a mini prep on overnight cultures of inoculated site direct mutagenesis tyrosinase from colonies #: 2, 4, 6 & 8. The method was according to the procedure described under Protocols.  
I performed a mini prep on overnight cultures of inoculated site direct mutagenesis tyrosinase from colonies #: 2, 4, 6 & 8. The method was according to the procedure described under Protocols.  
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==Fusionprotein of IgG protease & protein A (ZZ domain)==
 
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==Digestion of protein A ZZ domain in bank vector C==
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===Fusionprotein of IgG protease & protein A (ZZ domain)===
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[[Image:Bild22.jpg|280px]]
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===Digestion of protein A ZZ domain in bank vector C===
*DNA vector 2 ul
*DNA vector 2 ul
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*Restriction enzyme AgeI 1 ul
*Restriction enzyme AgeI 1 ul
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==Digestion of IgG protease in bank vector C==
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===Digestion of IgG protease in bank vector C===
This construct has previously been cut with the restriction enzyme NgoMIV (9/8-2010)and undergone a gel clean up.
This construct has previously been cut with the restriction enzyme NgoMIV (9/8-2010)and undergone a gel clean up.
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Incubated both digest samples in 37 °C for 5 min.
Incubated both digest samples in 37 °C for 5 min.
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==Ligation of the digest samples==
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===Ligation of the digest samples===
I made two versions of the ligation: # 1 & 2.  
I made two versions of the ligation: # 1 & 2.  
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Incubated both ligate samples in RT for 15 min.
Incubated both ligate samples in RT for 15 min.
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==Transform the ligate samples==
+
===Transform the ligate samples===
I transformed both of the ligated samples in each 100 ul Top 10 cells. The transformation method is according to the procedure decribed in protocols. However in the step 1 I thawed for 15 min instead of 10 min. In step 2 I added 3 ul of DNA, in step 6 I used LB instead of SOC and finally in step 10 I spread 100 ul of sample on a chloramphenicol plate.
I transformed both of the ligated samples in each 100 ul Top 10 cells. The transformation method is according to the procedure decribed in protocols. However in the step 1 I thawed for 15 min instead of 10 min. In step 2 I added 3 ul of DNA, in step 6 I used LB instead of SOC and finally in step 10 I spread 100 ul of sample on a chloramphenicol plate.
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==Glycerol stock on protein A (ZZ domain)==
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 +
===Glycerol stock on protein A (ZZ domain)===
I made a glycerol stock on protein A ZZ domain inserted in the bank vector with chloramphenicol.  
I made a glycerol stock on protein A ZZ domain inserted in the bank vector with chloramphenicol.  
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*400 ul Glycerol  
*400 ul Glycerol  
*800 ul Bacterial overnight sample
*800 ul Bacterial overnight sample
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 +
{{Stockholm/Footer}}

Latest revision as of 10:51, 26 October 2010


Contents

Nina

Mini prep on Tyrosinase

I performed a mini prep on overnight cultures of inoculated site direct mutagenesis tyrosinase from colonies #: 2, 4, 6 & 8. The method was according to the procedure described under Protocols.

spectrophotometer:

Tabelln.jpg


Fusionprotein of IgG protease & protein A (ZZ domain)

Bild22.jpg

Digestion of protein A ZZ domain in bank vector C

  • DNA vector 2 ul
  • H2O 14 ul
  • 10X fast digest buffer 2 ul
  • Restriction enzyme SpeI 1 ul
  • Restriction enzyme AgeI 1 ul

Digestion of IgG protease in bank vector C

This construct has previously been cut with the restriction enzyme NgoMIV (9/8-2010)and undergone a gel clean up.

  • DNA vector 2 ul
  • H2O 15 ul
  • 10X fast digest buffer 2 ul
  • Restriction enzyme SpeI 1 ul

Incubated both digest samples in 37 °C for 5 min.

Ligation of the digest samples

I made two versions of the ligation: # 1 & 2.

  1. 1:
  • IgG vector 1 ul
  • protein A gene 1 ul
  • quick ligase 1 ul
  • 2X ligase buffer 2.5 ul
  1. 2:
  • IgG vector 1.5 ul
  • protein A gene 2 ul
  • quick ligase 1 ul
  • 2X ligase buffer 3 ul

Incubated both ligate samples in RT for 15 min.

Transform the ligate samples

I transformed both of the ligated samples in each 100 ul Top 10 cells. The transformation method is according to the procedure decribed in protocols. However in the step 1 I thawed for 15 min instead of 10 min. In step 2 I added 3 ul of DNA, in step 6 I used LB instead of SOC and finally in step 10 I spread 100 ul of sample on a chloramphenicol plate.


Glycerol stock on protein A (ZZ domain)

I made a glycerol stock on protein A ZZ domain inserted in the bank vector with chloramphenicol.

Glycerol stock:

  • 400 ul Glycerol
  • 800 ul Bacterial overnight sample





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