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Team:DTU-Denmark/Notebook/24 July 2013
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decided to purify RFP in pZA21 without promoter as well as HAO, AMO and cycAX | decided to purify RFP in pZA21 without promoter as well as HAO, AMO and cycAX | ||
Revision as of 15:40, 24 July 2013
Contents |
208
Main purpose
Who was in the lab
Henrike, Julia, Gosia, Krystian
Procedure
USER reaction and transformation
We perform USER reaction, samples are as follows:
- plasmid pZA21 and AMO
- plasmid pZA21 and HAO
- negative control with water instead of insert
Reaction is performed in the same way as on 18-07-2013 with increased amount of insert (up to 14 uL due to low DNA concentration).
Restriction analysis
From last week transformants with AMO and HAO in pZA21 we performed plasmid isolation.
We perform restriction analysis with EcoRI. Expected fragments are as follows:
- For pZA21 with AMO:
3309 pz, 2283 pz
- For pZA21 with HAO:
2233 pz, 2124 pz, 930 pz
Primer diluting
PCR to extract Nir from Pseudosomonas with new USER primers
Results
first gel
decided to purify RFP in pZA21 without promoter as well as HAO, AMO and cycAX
purification gel
loaded the complete PCR reaction (~45 uL), cut out the bands of our products and used QIAgen gel extraction kit
- 1: 1 kb ladder
- 2: HAO
- 3: AMO
- 4: cycAX
- 5: RPF in pZA21 without promoter
- 6: RPF in pZA21 without promoter
- 7: RPF in pZA21 without promoter
gel for restriction analysis
Conclusion: We only get one band for each plasmid, so the inserts are not present.
Conclusion
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