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Main purpose

• Miniprep

Who was in the lab

Gosia, Henrike

Procedure

• miniprep of RFP in pZA21 and cycAX in pZA21

• made gycerol stocks from ON cultures supposedly containing RFP in pZA21 and cycAX in pZA21

• gel of yesterday's PCR (cycAX and Nir with USER primers)

• purified AMO for USER from gel

• PCR to add His-tags to construct 1 (Sec-GFP-RFP) and construct 2 (TAT-GFP-RFP)

• gel on today's PCR -> construct 1 has no product, construct 2 has an unkown product with length 900 bp

Plasmid isolation pZA21 with RFP and pZA21 with cycAX from transformants

According to standard protocol attached to GenElute™ Plasmid Miniprep Kit.

PCR

• cycAX fragment amplified with USER primers, template - isolated plasmid from colony 12 (orange transformant)

We performed PCR reaction in order to check the presence of cycAX insert in pZA21 plasmid because colony PCR for this fragment showed no positive results. We based on standard PCR programm with annealing temperature of 57°C and extension time of 1:30 min.

• Nir amplified with USER primers, template - cells of Pseudosomonas europea, 2 different PCR programms were used

We based on standard PCR programm and used annealing temperature of 62°C and extension time of 9 min for samples 1,2 and 4 min for samples 3,4,5.

AMO gel purification

PCR product was loaded on a gel in order to perform isolation from gel a fragment of desired lenght. It was performed according to procedure in kit ........ Qiagen.

Conclusion

Results

1% agarose gel of today's PCR