Team:HokkaidoU Japan/Notebook/aggregation Week 1
From 2012.igem.org
Contents |
July 2nd
On your mark...
July 3rd
Get set...
July 4th
Transformation
Transformation of BBa_B0015(dT), B0034(RBS), I179005(pT7), K346007(Ag43), and K542009(pLacI-RBS-Ag43) to DH5α
- Mixed 1 ul DNA solution with DH5α competent cells and incubated on ice in 30 min.
- Plated them on LBA(dt,RBS,T7) and LBC(Ag43, pLacI-RBS-Ag43). ]
July 5th
Transformation
K346007(Ag43) failed to produce colonies on LBC plate. Transformation of K346007(Ag43) to DH5α.
- Mixed 1 ul DNA solution with DH5α competent cells and incubated on ice for 30 min.
- To gain chrolamphenicol resistance solution was pre-cultivated for 2 hrs.
- Incubated on LBC for 21 hrs.
Single colony isolation
Performed single colony isolation of BBa_B0015, B0034, I179005 and K542009.
- Incubated the plates for 14 hrs and 30 mins
July 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 resuspended in 1 ml LB (A or C), and other colony was resuspended in 2 ml LB(A or C). 1 ml is for glycerol stocks and 2 ml is for mini-prep.
- 16 hrs Cultivation.
Single colony isolation
- Single colony isolation of K346007(Ag43) on LBC.
July 7th
Liquid culture
Liquid culture in LBC(Ag43).
- Resuspended two colonies from each plates.
- Both of colonies were dipped in 2 ml LBC, and then we cultivate them in 38C.
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. We used FastGene Plasmid Mini Kit(Nippon Gene)
- Got 50 ul of DNA solutions.
Glycerol stock
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) 1 ml in 16 hrs 30 min.
- 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.
- Added EtBr into TBE buffer and started Pre-migration.
- Migrated 1.2 ul of DNA solutions (1 ul is mini-prep products and 0.2 ul is Loading Dye) in 35 min.
- 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 digestion mix.
- I719005(40 ng/ul)
DNA solution | 12.5 ul |
EcoRI | 1 ul |
SpeI | 1 ul |
10xH Buffer | 3 ul |
DW | 12.5 ul |
- B0034(40 ng/ul)
DNA solution | 12.5 ul |
XbaI | 1 ul |
PstI | 1 ul |
10xM Buffer | 3 ul |
DW | 12.5 ul |
- pSB1K3(25 ng/ul)
DNA solution | 12 ul |
EcoRI | 1 ul |
PstI | 1 ul |
10xH Buffer | 3 ul |
DW | 13 ul |
Ethanol precipitation
For rising concentration of DNA solution which use for Ligation and removing restriction enzyme.
- Added 3 ul of NaoAc, 1.5 ul of glycogen and 75 ul of 100% ethanol.
- Centrifuged in 14000 rpm, 30 min at 4C.
- Remove supernatant and added 220ul of 70% ethanol.
- Centrifuged in 15000 rpm, 15 min at 4C.
- Remove supernatant and air drying in room temperature then added 10 ul of DW.
Ligation
All DNA solutions were digested. 3A assembly protocol required Ligation reaction should be in total 25 ul solution.
Ligation Mighty Mix | 12.5 ul |
pT7 | 2 ul |
RBS | 2 ul |
pSB1K3 | 2 ul |
DW | 6.5 ul |
Total | 25 ul |
Ligation reaction recipe was written below.
Degree | Minute |
16 | 30 |
65 | 10 |
4 | Hold |
ligation was finished.
But now pm10:00. 2.5hrs needs for doing transformation. Transformation would finish at am:0:30.
Withdraw!!!!
July 8th
- (pT7 + RBS)
Transformation
Transformation for pT7+RBS+pSB1K3
- Added DNA soltions (Ligation products) 1 ul to DH5α compitent cell.
- Stood on ice in 30 min.
- Added 600 ul of LB to transformed DH5α solution.
- Pre-cultivate in 2 hrs
- Splead 300 ul of LB&DH5α solution to LBK.
- Cultivated
- K346007(Ag43)
mini-prep
mini-prep for Liquid culture product of K346007(Ag43)
- Used FastGene Plasmid Mini Kit(Nippon Genetics)
- Elutioned in 50 ul
- First we eluted in colection tube. then moved in microcentrifuge tube.
Erectrophoresis
Erectrophoresis for mini-prep product(Ag43).
- Prepared 1% Agalose gel and added EtBr then pre-migration in 30 min.
- 1 ul 1kb ladder, 1.2 ul mini-prep product(1 ul is DNA solution and 0.2 ul is loading dye) added then migtrated.
Glycerol stock
Made glycerol stock of K346007 (Ag43).
- Parts written above were cultivated in LBC.
- Added glycerol and freezed at -80C
- (Ag43 + dT)
Assembling K346007(Ag43) + B0015(dT) with 2-piece assembly(Biobrisk standard assembly)
Digestion
Digested Ag43 and dT in solution by recipes Written below. Insert DNA required too much weight and volume(volume was calculated from concentration of DNA mini-prep product)from our calculation. There are no insurance of succession of digestion.
- Ag43(Insert)
DNA solution | 48 ul |
EcoRI | 1 ul |
SpeI | 1 ul |
10xH buffer | 6 ul |
4 ul | |
Total | 60 ul |
- dT(Vector)
3318bp(Ag43 + pSB1AK3)
DNA solution | 8 ul |
EcoRI | 1 ul |
XbaI | 1 ul |
10xM buffer | 2 ul |
DW | 8 ul |
Total | 20 ul |
K346007(Ag43) was 5190bp before digestion (Biobrick prefix + Ag43 + Biobrick suffix + pSB1C3). After digestion, Ag43 and pSB1C3 was separated and became fragments about 3120bp(Ag43) and 2070bp(pSB1C3). Gel image above shows there are two fragments, one fragment is about 2000bp other fragment is 3000bp. Digestion would be succeeded. About Vector(Boo15:dT), the DNA was circular so DNA migrated more far than Linear DNA. d+ (Circular DNA) migrated little more far than d- (Linear DNA). so we think digestion was succeeded.
Ethanol precipitation
Ethanol precipitation for gel extraction products(K346007(Ag43) and B0015(dT))
- Added 5 ul of NaoAc , 1.5 ul of glycogen and 125 ul of 100% ethanol to 50 ul DNA solutions.
- Centrifuged in 15000 rpm, 10min at 4C.
- Remove supernatant and added 220 ul of 70% ethanol.
- Centrifuged in 15000 rpm, 5 min at 4C.
- Remove supernatant and air drying in room temperature then added 10 ul of DW.
Ligation
All DNA solutions were digested. Used Ligation Mighty Mix(TakaraBio)
Ligation Mighty Mix | 5 ul |
Insert: Ag43 | 2 ul |
Vector: dT | 2 ul |
DW | 1 ul |
Total | 10 ul |
Ligation reaction recipe is following.
Degree | Minute |
16 | 30 |
65 | 10 |
4 | Hold |
Electrophoresis
Confirmation of succession of ligation.
- Prepared 1% Agalose gel and added EtBr then pre-migration in 30 min.
- Added 1kb ladder, Ligation product(1 ul) and digestion products (control:each solutions 1 ul).
- Migtrated in 30 min.
Transformation
Transformation for K346007(Ag43)+B0015(dT) on pSB1AK3.
- Added DNA soltions (Ligation products) 1 ul to DH5α compitent cell.
- Stood on ice in 30 min.
- Added 600 ul of LB to transformed DH5α solution.
- From 700 solution(100 ul is DH5α and 600 ul is LB), 100 ul add to 900 ul of LB(x10 solution)
- Spread 300 ul from 600(700-100) ul and 1000 ul of LB&DH5α solution to each LBA plates.
- Cultivated.