7.31
l ice breaker and self introductions
l laboratory safety training
8.1
l preparing to construct plasmid pTRIP with pTrac99k+RIP
¡ make nucleic acid gel
¡ PCR, gel electrophoresis, and DNA gel extraction for both vector and target gene
n failed -> multiple trials with different annealing temperatures
l preparing both liquid and solid LB culture medium
8.2
l constructing plasmid pTRIP
¡ Test the DNA concentration with Nanodrop
¡ homologous recombination of pTrac99k and RIP to obtain plasmid pTRIP
¡ pTRIP transformed into DH5α
8.3
l run another gel electrophoresis of pTRIP
l Send the company to sequencing
l EGFP and cddB ,pTRIP-E and pTRIP-C to varify the lengths
¡ DNA gel extraction of the correct strips
l tranform pTRIP-EGFP into DH5α and pTRIP-ccdB into DB3.1
l plating of DH5α-pTRIP-EGFP and DB3.1-pTRIP-ccdB
l leave the samples in the isothermic incubator at 37°C overnight
8.4
l Monoclonal antibodies were selected and verified, and put into kanamycin-included LB medium, incubate at 37°C, shaking for 12-16h
8.5
l plasmid extraction of bacterial fluids
¡ test the concentration with Nanodrop
¡ gel electrophoresis for varification
¡ Send the company to sequencing
l inducing protein expression
l prepare protein gel for SDS-PAGE using Sangon 12.5% SDS-PAGE Color Preparation kit
8.6
l Ultrasonic crushing, protein concentration(pTRIP-EGFP and pTRIP-ccdB)
8.7
l protein extraction
l SDS-PAGE (pTRIP-EGFP and pTRIP-ccdB)
8.8
l The fluorescence intensity of EGFP was measured
l The reporter gene EGFP was observed under microscope
8.9
l The pTRIP-ccdB was transformed into E.coil DH5α and BL21 ( DE3 )
8.10
l Monoclonal antibody verification of pTRIP-ccdB in E.coil DH5α and BL21 ( DE3 )
8.11
l Detection of pTRIP-ccdB growth ability in E.coil DH5α and BL21 ( DE3 )