Team:CBNU-Korea/Notebook
From 2010.igem.org
You can write a background of your team here. Give us a background of your team, the members, etc. Or tell us more about something of your choosing. | |
Tell us more about your project. Give us background. Use this is the abstract of your project. Be descriptive but concise (1-2 paragraphs) | |
Team Example |
Home | Team | Official Team Profile | Project | Parts Submitted to the Registry | Modeling | Notebook | Safety |
---|
5/7
Membership meeting
New participant of CBNU-KOREA team.
Introduced what is the iGEM, what we should do if we join the competition.
5/15
Concept meeting
Decided 2010 CBNU-KOREA iGEM team’s project(almost 4hr)
5/18
Researching
Finding related articles, paper and so on.
Essential gene data search
5/25
Strategy about database
Planned how to re-construct essential gene database.
7/19
Plasmid Culture
• Pick up a single colony from fresh cultured LB agar plate.
• Our established media and inoculate the cell into the 5ml of fresh LB liquid media (contains antibiotics) and our established media at 37℃ with shaking for 12hr.
• plasmid : pSB1C3, pSB1A3, pSB1T3, pSB1K3 (contain J54450)
7/20
Plasmid extract
• Uses kit and extracted plasmid. (pSB1C3, pSB1A3, pSB1T3, pSB1K3)
• Confirmed size by electrophoresis.
7/23
Plasmid Digest
• Digest of 4 plasmid.
• Used EcoRⅠ-PstⅠ restriction endonuclease.
• Confirmed size by electrophoresis.
7/24
Primer Design
• Designed Vibrio cholerae's oriC2vc, parA, parB, parS, dif gene primer using ‘Ginko primer’.
7/28
DNA Amplification
• Amplified ori, parA, parB, parS, dif gene of vibrio cholerae.
• Confirmed size by electrophoresis.
Concentration
• Because concentration of parS, dif gene was low, it was hard to confirm result by electrophoresis.
• Concentrated gene sample
7/29
DNA(gene) Digest
• Digest of 4 plasmid.
• Used EcoRⅠ-PstⅠ restriction endonuclease.
• Confirmed size by electrophoresis.
7/30
Prepared Compentent cell
• E.coli DH10B, DH5α, JM109
8/2
DNA Ligation/Transformation
• Ligation inserting gene in vector
(parA+pSB1A3, parS+pSB1C3, ori+pSB1K3, dif+pSB1A3)
• Transformation (Competent cell : E.coli DH10B)
• Spreading on plate (each antibiotics contained LB agar media)
8/3
Cell Culture
• Pick up a single colony from fresh cultured LB agar plate.
• Our established media and inoculate the cell into the 5ml of fresh LB liquid media (contains antibiotics) and our established media at 37℃ with shaking for 12hr.
• vector : parA+pSB1A3, parS+pSB1C3, ori+pSB1K3, dif+pSB1A3
8/4
Vector extract
• Uses kit and extracted plasmid.
(parA+pSB1A3, parS+pSB1C3, ori+pSB1K3, dif+pSB1A3)
• Confirmed size by electrophoresis.
8/9
Plasmid Digest
• Digest of 4 vector.
• ori+pSB1K3, parA+pSB1A3, parS+pSB1C3 digests by EcoRⅠ-SpeⅠ restriction endonuclease and dif+pSB1A3 gene digested by XbaⅠ-PstⅠ restriction endonuclease.
• Confirmed size by electrophoresis.
• Came out as size of ori+pSB1K3 gene is smaller.
8/10
Sequencing Order
• Analyzed plasmid DNA extraction (cultured cell)
8/16
Primer Again design
• According to analysis result, primer of ori gene knew wrong fact.
• Designed primer of ori gene again.
• Primer of I51020 and p1003 designed.
8/20
DNA Amplification
• Amplified I51020, p1003
• Confirmed size by electrophoresis.
8/23
Plasmid Digest
• I51020 digests by EcoRⅠ-PstⅠ restriction endonuclease.
• p1003 digests by XbaⅠ-PstⅠ restriction endonuclease.
• Confirmed size by electrophoresis.
8/24
DNA Ligation/Transformation
• Ligated inserting gene in vector
(Inserted ori gene and p1003 antibiotic resistance cassette in I51020 vector, inserted parS gene and dif gene in pSB1A3 vector)
• Transformation (Competent cell : E.coli DH10B)
• Spreading on plate (each antibiotics contained LB agar media)