Cloning of the genes that code for three major subunits of Escherichia coli polymerase III Chengxi...
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Transcript of Cloning of the genes that code for three major subunits of Escherichia coli polymerase III Chengxi...
Cloning of the genes that code for three major subunits of Escherichia coli polymerase III
Chengxi ShiMolecular Biotechnology and
BioinformaticsUppsala University
winter,2005
About Research Training
Period: November to December Institute: Department of Cell & Molecular Biology
Work time: 9 am to 5 pm at weekdays
Supervisor: Prof. Gerhart Wargner
Department Information
The department is divided into 6 programs
Mikrobiologi Gerhart’s research focus on: ’riboregulator’
regulatory RNAs in bacteria
Observation: mutates all the genes that are know
to negatively control replication
DNA content only goes up 1.5 – 2 folds
A thought: the number of DNA polymerase molecules in the cell is limiting (usually 8-10 molecules per cell)
Strategy
Use pBAD-TOPO vector to insert in order dnaE, dnaQ, holE, and with very little spacing inbtween.
PCR out the genes the primers should carry different restriction sites
PCR out the genes the primers should carry different restriction sites
Overview design primers re-streak bacteria stain
PCR plasmid miniprep
PCR product purification
enzyme cleavage enzyme cleavage
gel extraction gel extraction
ligation
transform into competent cells
colony PCR test
Primer designing For each insert: Include translation initiation site ATG
For the first insert: Include the Shine-Dalgarno sequence
GGAA
dnaE forward: 5′- [P] – CTGACTGCAGGGAATCTGAAGATGTCTGAA
PstI dnaE reverse: 5′- TAGAATTCTACCATGGTTAGTCAAACTCCAGTTCCA
EcoRI NcoI
dnaQ forward: 5′- TACCATGGAAGTCTGACATAAATGACCGCT
NcoI
dnaQ reverse: 5′- TAGAATTCTAGGTACCTTATGCTCGCCAGAGGCAAC
EcoRI KpnI
holE forward: 5′- TAGGTACCGAGGAGATTAAGAATG
KpnI
holE reverse: 5′- TAGAATTCTTATTTAAGTTTGGGCT
EcoRI
Acknowledgement
Many thanks to Prof. Gerhart Also thank the member of the lab,
especially Klas, Cia, Shiying and Erik