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Team:Stockholm/14 August 2010
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NinaSchiller (Talk | contribs) (→Transform the ligate samples) |
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===Fusionprotein of IgG protease & protein A (ZZ domain)=== | ===Fusionprotein of IgG protease & protein A (ZZ domain)=== | ||
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===Digestion of protein A ZZ domain in bank vector C=== | ===Digestion of protein A ZZ domain in bank vector C=== | ||
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| - | ==Glycerol stock on protein A (ZZ domain)== | + | ===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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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:
Fusionprotein of IgG protease & protein A (ZZ domain)
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:
- IgG vector 1 ul
- protein A gene 1 ul
- quick ligase 1 ul
- 2X ligase buffer 2.5 ul
- 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
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