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Main purpose

• Miniprep of biobrick transformants pSB1T3, pSB3T5, pSB1AK3, pSB1A3 from 10-07-2013
• gel analysis of the colony PCRs
• PCR on AMO with USER-primers with lower temperature to see if we can get any product at all
• Order better primers for colony PCR
• PCR to extract the AMO with USER primers using Nitrosomonas cells as a template

Who was in the lab

Kristian, Gosia, Henrike, Julia

Procedure

• Plasmid isolation as described in the sigma MiniPrep kit.

• PCR in order to amplify AMO gene using USER primers and 2uL of liquid culture of Nitrosomonas europea. Primers 17a and 17b were used and master mix for 5 samples was prepared. PCR program was the same as on 12-07-2013 for AMO.

• innoculation of ON cultures of colony 12 of cycAX and colonies 3, 4 and 5 of RFP

Results

• gel for the colony PCR is empty but some of the RFP containing colonies are red and fluoresce and colony 12 of the cycAX containing E. coli is visibly orange

Conclusion

Nothing was on the colony-PCR gel and we speculate that this is because the USER-primers used to this are not very good to this purpose. We therefore ordered new primers for colony PCR which contain no uracil. The new primers can be used with PHUSION polymerase or any other cheaper alternative.

1 colony (number 12) among transformants with pZA21 and cytochromes (cycAX) changed color for orange, probably it is because of the presence of cytochromes. We will isolate plasmid from this colony and check if the insert is present by restriction analysis. Also all colonies from plate transformed with pZA21 and RFP turned pink and show red color exposed to UV light. Colony 3,4 and 5 from RFP in pZA21 transformants were chsen to inoculate liquid medium in order to isolate plasmids.

We will make glycerol stocks from chosen colonies in liquid cultures tomorrow.

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