7.31

ice breaker and self introductions

laboratory safety training

 

8.1

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

failed -> multiple trials with different annealing temperatures

preparing both liquid and solid LB culture medium

 

8.2

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

run another gel electrophoresis of pTRIP

Send the company to sequencing

EGFP and cddB ,pTRIP-E and pTRIP-C to varify the lengths

¡ DNA gel extraction of the correct strips

tranform pTRIP-EGFP into DH5α and pTRIP-ccdB into DB3.1

plating of DH5α-pTRIP-EGFP and DB3.1-pTRIP-ccdB

leave the samples in the isothermic incubator at 37°C overnight

 

 

8.4

Monoclonal antibodies were selected and verified, and put into kanamycin-included LB medium, incubate at 37°C, shaking for 12-16h

 

8.5

plasmid extraction of bacterial fluids

¡ test the concentration with Nanodrop

¡ gel electrophoresis for varification

¡ Send the company to sequencing

inducing protein expression

prepare protein gel for SDS-PAGE using Sangon 12.5% SDS-PAGE Color Preparation kit

 

8.6

Ultrasonic crushing, protein concentration(pTRIP-EGFP and pTRIP-ccdB)

 

8.7

protein extraction

SDS-PAGE (pTRIP-EGFP and pTRIP-ccdB)

 

8.8

The fluorescence intensity of EGFP was measured

The reporter gene EGFP was observed under microscope

 

8.9

 The pTRIP-ccdB was transformed into E.coil DH5α and BL21 ( DE3 )

 

8.10

Monoclonal antibody verification of pTRIP-ccdB in E.coil DH5α and BL21 ( DE3 )

 

8.11

Detection of pTRIP-ccdB growth ability in E.coil DH5α and BL21 ( DE3 )