Team:HokkaidoU Japan/Notebook

From 2012.igem.org

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Revision as of 09:45, 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!!


Experiment Calender

July
S M T W T F S
1 2 3 4 5 6 7
8 9 10 11 12 13 14
15 16 17 18 19 20 21
22 23 24 25 26 27 28
29 30 31  


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

  1. Added each DNA solutions (1ul) into DH5α comeptent cell and standed on ice in 30 min.
  2. 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.

  1. Add DNA solution(1ul) into DH5α comeptent cell and stand on ice in 30 min.
  2. Pre-cultivated in 2hrs.
  3. Cultivated on LBC in 21hrs.
Single colony isolation

Single colony isolation of BBa_B0015, B0034, I179005 and K542009.

  1. Picked up one colony.
  2. 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)

  1. Picked up two colonies from each plates.
  2. 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.
  3. 16hrs Cultivation


Single colony isolation
  1. 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
  1. mini-prep of dT,RBS,pT7 and pLacI-RBS-Ag43.
  2. Elution in 50ul buffer


Glycerol stock

Made glycerol stocks of dT,RBS,pT7 and pLacI-RBS-Ag43.

  1. Parts written above were cultivated in LBA(dT,RBS,pT7) and LBC(pLacI-RBS-Ag43) 1ml in 16hrs 30min.
  2. Add glycerol and Freeze at -80C


120707 I719005 B0034 B0015 K542009 mini-prep umeuchi.jpg

Electrophoresis

Electrophoresis to predict concentration of mini-prep products(dT,RBS,pT7 and pLacI-RBS-Ag43).

  1. Used 1% agarose gel.
  2. Pre-migration.
  3. Migrated 1.2ul of DNA solutions (1ul is mini-prep products and 0.2ul is Loading Dye) in 35min.
  4. 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
PstI 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.

  1. Added 3ul of NaoAC, 1.5ul of glycogen and 75ul of 100% ethanol.
  2. Centrifuged in 14000rpm, 30min at 4C.
  3. Remove supernatant and added 220ul of 70% ethanol.
  4. Centrifuged in 15000rpm, 15min at 4C.
  5. Remove supernatant and air drying in room temperature then added 10ul of DW.


Ligation


Transformation


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