Lab 208

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

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 aeruginosa, 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 Qiagen Spin miniprep kit.

PCR to add His-tags to Hellow World constructs

performed PCR on the two constructs made in the Hello World project

used primers 19a, 19b for Sec-construct

used primers 20a, 20b for TAT-construct

program for Sec-construct: 60C annealing temperature and 4:00 elongation time

program for TAT-construct: 56C annealing temperature and 4:00 elongation time

Results

1% agarose gel of today's PCR of the constructs with his tags.

• 1: 1kb ladder
• 2: Sec-construct
• 3: Sec-construct
• 4: Sec-construct
• 5: TAT-construct
• 6: TAT-construct
• 7: TAT-construct

Conclusion

We obtained TAT construct!

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