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Expression plasmid design

 

In this block of the course you will learn (1) how to design and practically construct protein expression vectors and (2) how to assay the protein expression.

 

You will have to solve tasks of the computer lab exercises (see computer lab) and send your solutions to Anton before November 25th. The results of wet labs must be well documented in your lab books (the quality of your notes will be checked by Dee). No lab report this time.

 

Note: The studies will take place in Biocenter (BioC)

 

Nov 7th, Monday

10:00-12:00 First meeting. Students receive subjects of presentations, which they will have to prepare. BioC C216

 

 

Nov 8th, Tuesday

9:00-10:00 Lecture: An overview of E. coli expression systems Part 1 (Anton) BioC C216

10:00-12:00 Computer lab: Vector NTI tutorial (Anton) Bibliotekets Datorsal 2

12:00-14:00 Lunch

14:00-17:00 Computer lab: Vector NTI tutorial (Anton) KC Datorsal

 

 

Nov 9th, Wednesday

 9:00-10:00 Lecture: An overview of E. coli expression systems Part 2 (Anton) BioC C216

10:00-13:00 Computer lab: Design of expression constructs (Anton) Bibliotekets Datorsal 1

13:00-14:00 Lunch

14:00-17:00 Computer lab: Design of expression constructs (Anton) Bibliotekets Datorsal 2

 

 

Nov 10th, Thursday

9:00-10:00 Lecture: An overview of eukaryotic expression systems (Mats) BioC C216

Printed lecture slides can be obtained from Mats!

10:00-13:00 Computer lab: Design of expression constructs (Anton) Bibliotekets Datorsal 2

13:00-14:00 Lunch

14:00-17:00 Computer lab: Design of expression constructs (Anton) Bibliotekets Datorsal 2

 

 

Nov 11th, Friday

09:00-12:00 Computer lab: Design of expression constructs (Anton) Bibliotekets Datorsal 1

13:00-14:00 Lunch

14:00-17:00 Computer lab, Final Report: Design of expression constructs (Anton) Bibliotekets Datorsal 1

 

 

Nov 14th, Monday

Wet Lab (Dee, Roy is helping) BioC:Lab

9:00-11:00 PCR amplification of the gene sequence of the usher middle domain (UMD), casting an agarose gel

11:00-12:10 Separation of the PCR reaction mixture on the agarose gel. Lunch during the electrophoresis (1 hour).

12:20-12:50 Purification of the band of the PCR product from the agarose gel using a gel extraction of kit

13:00-13:30 TOPO cloning reaction

14:00-16:00 Transformation of TOP10 competent cells with the TOPO cloning reaction mixture

 

 

Nov 15th, Tuesday

 

Wet Lab (Dee) BioC:Lab

9:00-16:00 Colony-PCR:

  9:00-9.20 LB medium inoculation with selected colonies

     14:30-16:00 PCR

16:20-16:50 Purification of plasmids containing the insertion

17:00-17:30 Transformation of BL21(DE3) cells with the constructed plasmids

 

 

Nov 16th, Wednesday

9:00-16:00 Record obtaining results in the lab book (could be done at any location)

Wet Lab (Dee)

Some time at the end of the day Setting night cultures for the test expression

 

 

Nov 17th, Thursday

Wet Lab (Dee) BioC:Lab

9:00 Inoculation of cultures for the expression experiment

~ 10:30 Inducing the protein expression

~ 12:30 Harvesting the cells

13:00-14:00 Lunch

14:00 Sonication of cells

15:30-17:00 SDS gel

 

 

Nov 18th, Friday

9:00-16:00 Finish recording results in the lab book (could be done at any location)

 

 

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