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Team:HokkaidoU Japan/Notebook
From 2012.igem.org
(Difference between revisions)
| Line 74: | Line 74: | ||
*4th | *4th | ||
;Transformation | ;Transformation | ||
| - | + | Transformation of BBa_B0015(dT), B0034(RBS), I179005(pT7), K346007(Ag43), and K542009(pLacI-RBS-Ag43) in DH5α | |
| - | #Cultivated on LBA(dt,RBS,T7) and LBC(Ag43, pLacI-RBS-Ag43) . pT7 was 21hrs and Others were 20hrs cultivated | + | #Added each DNA solutions (1ul) into DH5α comeptent cell and standed on ice in 30 min. |
| + | #Cultivated on LBA(dt,RBS,T7) and LBC(Ag43, pLacI-RBS-Ag43). E.coli cultivate in LBC was pre-cultivated in 2 hrs. pT7 was 21hrs and Others were 20hrs cultivated | ||
<br> | <br> | ||
| Line 81: | Line 82: | ||
;Transformation | ;Transformation | ||
K346007(Ag43) was failed to cultivate on LBC plate. | K346007(Ag43) was failed to cultivate on LBC plate. | ||
| - | + | Transformation of K346007(Ag43) in DH5α. | |
| + | #Add DNA solution(1ul) into DH5α comeptent cell and stand on ice in 30 min. | ||
| + | #Pre-cultivated in 2hrs. | ||
#Cultivated on LBC in 21hrs. | #Cultivated on LBC in 21hrs. | ||
;Single colony isolation | ;Single colony isolation | ||
| - | + | Single colony isolation of BBa_B0015, B0034, I179005 and K542009. | |
| + | #Picked up one colony. | ||
#Cultivation on LBA(dt,RBS,T7) and LBC(pLacI-RBS-Ag43) in 14hrs30mins | #Cultivation on LBA(dt,RBS,T7) and LBC(pLacI-RBS-Ag43) in 14hrs30mins | ||
| - | BBa_K542009 was Ag43 only part! And the part didn't have Biobrick suffix. | + | BBa_K542009 was Ag43 only part! And the part didn't have Biobrick suffix. |
*6th | *6th | ||
;Liquid culture | ;Liquid culture | ||
| - | + | Liquid culture in LBA(dT,RBS,pT7) and LBC(pLacI-RBS-Ag43) | |
| - | # | + | #Picked up two colonies from each plates. |
| + | #One colony was dipped in 1ml LB (A or C), and other colony was dipped in 2ml LB(A or C). 1ml is for glycerol stocks and 2ml is for mini-prep. | ||
#16hrs Cultivation | #16hrs Cultivation | ||
| + | <br> | ||
;Single colony isolation | ;Single colony isolation | ||
#Single colony isolation of K346007(Ag43). | #Single colony isolation of K346007(Ag43). | ||
Revision as of 08:57, 7 July 2012
| Home | Team | Official Team Profile | Project | Parts Submitted to the Registry | Modeling | Notebook | Safety | Attributions |
|---|
Contents |
Hello
We are team HokkaidoU Japan! Today we learn and start to edit wiki.
(>ω<)
Dear Mr.Ortiz, I saw the help page which you edited.
hola!
March
Spring Boot Camp
- date
- March 5 (Mon) ~ March 9 (Fri)
Monday, March 5
- Session #1
- Short lecture about moleculer biology (Mr.Yamazaki, our adviser)
- Session #2
- Tutrial: How to use 'Unipro UGENE' (iTakeshi)
- Session #3
- Guidance: Wiki Reading (Laury)
- Example: 2010 MIT
Tuesday, March 6
- Session #4~6
- Reading Wikis in turn and discussions
- 2010 NYU
- 2009 Cambridge
- 2009 Growningen
Wednesday, March 7
- Session #7~11
- Reading Wikis (2)
- 2010 Washington
- 2009 Valencia
- 2011 Barklay
- 2010 Paris
- 2010 Bristol
Thursday, March 8
- Session #12
- 2012 Project Brainstorming
- The details is secret! :)
- Session #13
- Guidance: How to read papers (Laury)
Friday, March 9
- Session #14
- 2012 Project Brainstorming (2)
- Session #15
- Guidance: How to look up papers you want (Laury)
- Session #16
- Tutorial: Modeling the behavior of cells (iTakeshi)
- Session #17
- Final Session: Reviewing this camp
- Party!!
July
phaABC team
now experimenting...
Ag43&Lysis team
weak 1(4th~10th)
- 4th
- Transformation
Transformation of BBa_B0015(dT), B0034(RBS), I179005(pT7), K346007(Ag43), and K542009(pLacI-RBS-Ag43) in DH5α
- Added each DNA solutions (1ul) into DH5α comeptent cell and standed on ice in 30 min.
- Cultivated on LBA(dt,RBS,T7) and LBC(Ag43, pLacI-RBS-Ag43). E.coli cultivate in LBC was pre-cultivated in 2 hrs. pT7 was 21hrs and Others were 20hrs cultivated
- 5th
- Transformation
K346007(Ag43) was failed to cultivate on LBC plate. Transformation of K346007(Ag43) in DH5α.
- Add DNA solution(1ul) into DH5α comeptent cell and stand on ice in 30 min.
- Pre-cultivated in 2hrs.
- Cultivated on LBC in 21hrs.
- Single colony isolation
Single colony isolation of BBa_B0015, B0034, I179005 and K542009.
- Picked up one colony.
- Cultivation on LBA(dt,RBS,T7) and LBC(pLacI-RBS-Ag43) in 14hrs30mins
BBa_K542009 was Ag43 only part! And the part didn't have Biobrick suffix.
- 6th
- Liquid culture
Liquid culture in LBA(dT,RBS,pT7) and LBC(pLacI-RBS-Ag43)
- Picked up two colonies from each plates.
- One colony was dipped in 1ml LB (A or C), and other colony was dipped in 2ml LB(A or C). 1ml is for glycerol stocks and 2ml is for mini-prep.
- 16hrs Cultivation
- Single colony isolation
- Single colony isolation of K346007(Ag43).
- 7th
3A assembly! Assembled pT7, RBS and pSB1C3 by 3A assembly. This 3A assembly is our first try!
- mini-prep
- mini-prep of dT,RBS,pT7 and pLacI-RBS-Ag43.
- Elution in 50ul buffer
- Glycerol stock
Made glycerol stocks of dT,RBS,pT7 and pLacI-RBS-Ag43.
- Parts written above were cultivated in LBA(dT,RBS,pT7) and LBC(pLacI-RBS-Ag43) 1ml in 16hrs 30min.
- Add glycerol and Freeze at -80C
- Electrophoresis
Electrophoresis to predict concentration of mini-prep products(dT,RBS,pT7 and pLacI-RBS-Ag43).
- Used 1% agarose gel.
- Pre-migration.
- Migrated 1.2ul of DNA solutions (1ul is mini-prep products and 0.2ul is Loading Dye) in 35min.
- Took a photograph of 1% agarose gel that finished electrophoresis.
- Digestion
Digestion of I719005, B0034 and pSB1K3
Digestion recipe
All parts were reacted in 30ul solution.
- I719005(40ng/ul)
| DNA solution | 12.5ul |
| EcoRI | 1ul |
| SpeI | 1ul |
| 10xH Buffer | 3ul |
| DW | 12.5ul |
- B0034(40ng/ul)
| DNA solution | 12.5ul |
| XbaI | 1ul |
| SpeI | 1ul |
| 10xM Buffer | 3ul |
| DW | 12.5ul |
- pSB1K3(25ng/ul)
| DNA solution | 12ul |
| EcoRI | 1ul |
| PstI | 1ul |
| 10xH Buffer | 3ul |
| DW | 13ul |
- Ethanol precipitation
For rising concentration of DNA solution which use for Ligation and removing restriction enzyme.
- Added 3ul of NaoAC, 1.5ul of glycogen and 75ul of 100% ethanol.
- Centrifuged in 14000rpm, 30min at 4C.
- Remove supernatant and added 220ul of 70% ethanol.
- Centrifuged in 15000rpm, 15min at 4C.
- Remove supernatant and air drying in room temperature then added 10ul of DW.
- Ligation
- Transformation
