正交验证组试验记录-iGEM2018.PDFVIP

正交验证组实验记录 Date: 8.1 1. Use 10μ ddH2O to dilute 2018kit part Plate 6-9M T7-RFP Plate 3-8M T7-sfGFP -Terminator Plate 3-12I T7-GFP-Terminator 2. Pick 1μ part liquid transform DH5α and BL21 Tomorrow observe transformation result Date: 8.2 1. Observe transformation result: All plates have no colony, transformation fail. 2. Three part (6-9M, 3-8M, 3-12I) transform DH5α and BL21 again. Tomorrow observe transformation result Date: 8.3 1. Observe transformation, Result: All DH5α transformation succeed; BL21 has only RFP transformation succeeded (and the colony is red), sfGFP and GFP transformation fail. 2. Pick the successful transform result colony, culture in LB medium for 12h 3. Culture the bacterium in LB medium, result extract plasmid, agarose gel electrophoresis for verification. Agarose gel electrophoresis, result is consistent with, but because plasmid is superhelix, we can’t verify it. 4. Extracted plasmid sequenced, verify part Wait for tomorrow sequencing result 5. Store RFP in BL21 glycerin mixture Date: 8.4 Wait for sequencing result Date: 8.5 1. Observe sequencing result: SfGFP sequencing result is consistent with the plasmid map, but GFP suggests a point but mutation (wiki says the part potentially has point mutation. 2. Extracted sfGFP and GFP plasmid transform BL21 Tomorrow observe transformation result Date: 8.6 1. Observe transformation result: Transformation result is ok, and the colony is nice. 2. Transformation, pick the result colony and culture in LB medium. 3. Because RFP part has no terminator, which needs to be added a new terminator. Use enzyme digestion ligation, cleave from Biobrick Suffix, which needs to add enzyme digestion site (SpeI and PstI) at the frank of the terminator, design