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experimenter: LXL, CCY

plx007 precipitation and rescue

then ligate lacZa and plx007, also control as self-ligation of plx007

PCR SD A-H,Y,Cro-lacZa

 template: MG1655 Genome
 Primer F: SDA-H,Y,cro-lacZa-NotI-F
 Primer R: lacZa-XhoI-R

PKU Switch 07-7-12 PCR 1.jpg

left to right: A, B, C, D, E, 100bp ladder

PKU Switch 07-7-12 PCR 2.jpg

left to right: F, G, H, Y, cro, 100bp ladder

回收: A-cro loading 2uL

PKU Switch 07-7-12 回收.jpg

digestion overnight

fragment 12uL H buffer 10uL BSA 10uL Triton 10uL NotI 1uL Xho I 2uL H2O 22uL

miniprep and digestion test of SDD'-EGFP-pcc002

plasmid 7uL BamHI 0.5uL XhoI 0.5uL K 2uL H2O 10uL

PKU Switch 07-7-12 SDD'-EGFP-pcc002.jpg

SDD-EGFP-pcc002 colony PCR

primer: SDD-EGFP-BamHI-F/pcc-test-R

RD1, CD1-PMD18T colony PCR

Primer: M13F/M13R

PKU Switch 07-7-12 .jpg

colony PCR lacZa-plx007

primer: test-F/test-R positive control: lacZa self-ligation negative control: plx007 self-ligation

PKU Switch 07-7-12 lacZa-plx007 1.jpg

PKU Switch 07-7-12 lacZa-plx007 2.jpg

maybe there is a problem with the method of colony test PCR. We can use SDY-LacZa-NotI-F/lacZa-XhoI-R as a change.

==SDD_EGFP_pcc002 colony PCR region by region, 5 per region

PKU Switch 07-7-12 SDD EGFP pcc002.jpg

transformation of LacZa-plx007 abd plx007 self-ligation