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Main purpose

Make USER-reaction to get pZA21 with arabinose inducible promoter. Make PCR to get more HAO and AMO with USER-primers. Finish the restriction analysis.

Who was in the lab

Henrike, Julia, Gosia, Kristian

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).

With the same procedure we also preformed a USER reaction with pZA21::RFP PCR-amplified with no promoter and araBAD from BBa_K808000.

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

Tubes have 4 labels (pZA21 only has 3), in vertical order:

• 'Nir' or 'pZ', tells which product is being made
• '1' - part 1 of Nir OR '2' - part 2 of Nir OR 'w' - whole Nir (on the pZ tubes 1 and 2 are just duplicates)
• '5' - used 5uL of N.europeae culture as template OR '10' - used 10uL of N.europeae culture as template
• 'ex' - extraction PCR, using the new non-USER primers that Jakob made OR 'U' - using the new USER primers that Jakob made

two programs:

all extraction PCRs and part 2 of Nir with USER primers - 54C, 5:00

part 1 of Nir with USER primers and pZA21 for ligation with Nir - 50C, 5:00

PCR with USER-primers on HAO and AMO

Standard USER-PCR was made with the HAO and AMO templates purified earlier today. The reactions where done in duplicates and with 2 different concentrations:

• HAO 5 is with 5uL template
• HAO 10 is with 10uL template
• AMO 5 is with 5uL template
• AMO 10 is with 10uL template

All tubes where run with 52C annealing temperature and 3 min extension time.

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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