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From 2013.igem.org

Contents 1 208 1.1 Main purpose 1.2 Who was in the lab 1.3 Procedure 1.3.1 USER reaction and transformation 1.3.2 Restriction analysis 1.3.3 Primer diluting 1.3.4 PCR to extract Nir from Pseudosomonas with new USER primers 1.4 Results 1.5 first gel 1.6 purification gel 1.7 gel for restriction analysis 1.8 Conclusion

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Main purpose

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Who was in the lab

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Henrike, Julia, Gosia, Krystian

Procedure

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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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