Team:HokkaidoU Japan/Notebook/aggregation Week 1

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Transformation

Transformation of BBa_B0015(dT), B0034(RBS), I179005(pT7), K346007(Ag43), and K542009(pLacI-RBS-Ag43) into DH5α

1. Mixed 1 ul DNA solution with DH5α competent cells and incubated on ice for 30 min.
2. Plated them on LBA(dt,RBS,T7) and LBC(Ag43, pLacI-RBS-Ag43). ]

To gain chloramphenicol resistances transformed cell solution was incubated for 2 hrs. pT7 plate was incubated for 21 hrs and Others for 20 hrs.

Transformation

K346007(Ag43) failed to grow on LBC plate. Transformation of K346007(Ag43) into DH5α.

1. Mixed 1 ul DNA solution with DH5α competent cells and incubated on ice for 30 min.
2. To gain chrolamphenicol resistance solution was pre-incubated for 2 hrs.
3. Incubated on LBC for 21 hrs.

Single colony isolation

Performed single colony isolation of BBa_B0015, B0034, I179005 and K542009.

1. Incubated the plates for 14 hrs and 30 mins

After consulting part page we found out that BBa_K542009 was Ag43 basic part, not composite! And the part didn't have Biobrick suffix.

Incubation for plasmid extraction and glycerol stock

Incubated in LBA(dT,RBS,pT7) and LBC(pLacI-RBS-Ag43) solution.

1. Picked up two colonies from each plate.
2. First colony was re-suspended in 1 ml LB (A and C respectively) for glycerol stock. Second colony was re-suspended in 2 ml LB(A and C respectively) for plasmid extraction.
3. Incubated for 16 hrs.

Single colony isolation

1. Single colony isolation of K346007(Ag43) on LBC.

Incubation of plasmid extraction and glycerol stock

Incubation of (Ag43) in LBC liquid medium.
Two colonies were re-suspended in 2 ml LBC. We incubated them at 38C.
One of the tubes was incubated for 8 hrs. It's for glycerol stock.

3A assembly!!!

Assembled pT7, RBS and pSB1C3 by 3A assembly. This is our first try!

Plasmid extraction

1. Plasmid extraction of dT,RBS,pT7 and pLacI-RBS-Ag43. We used FastGene Plasmid Mini Kit(Nippon Gene)
2. We got 50 ul of DNA solutions.

Glycerol stock

Glycerol stocks of dT,RBS,pT7 and pLacI-RBS-Ag43.

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

Estimation of plasmid concentration

Electrophoresis to estimate the concentration of plasmid extraction products(dT,RBS,pT7 and pLacI-RBS-Ag43).

1. Used 1% agarose gel.
2. Added EtBr to TBE buffer and soaked the gel by applying current.
3. Ran 1.2 ul of DNA solutions (1 ul is plasmid extraction product and 0.2 ul is Loading Dye) for 35 min.
4. Took a photograph of 1% agarose gel.

Digestion of I719005, B0034 and pSB1K3

Digestion reaction All parts were digested in 30 ul reaction solution.

• 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 of Digestion products

Concentrating the DNA solution which and removing restriction enzymes.

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

Ligation (3A Assembly)

3A assembly requires Ligation reaction total volume to be 25 ul.

Ligation Mighty Mix	12.5 ul
pT7	2 ul
RBS	2 ul
pSB1K3	2 ul
DW	6.5 ul
Total	25 ul

Incubation of ligation reaction.

Degree	Minute
16	30
65	10
4	Hold

ligation was finished. But now is 10 pm. 2.5hrs are needed for transformation. Transformation would be finished at 0:30 am.

Withdraw!!!!

• (pT7 + RBS)

Transformation

Transformation for pT7+RBS+pSB1K3

1. Added DNA soltions (Ligation products) 1 ul to DH5α compitent cell.
2. Stood on ice for 30 min.
3. Added 600 ul of LB to transformed DH5α solution.
4. Pre-incubated for 2 hrs at 37C.
5. Splead 300 ul of LB&DH5α solution to LBK.
6. Incubated for 19 hrs.

Plasmid extraction

Plasmid extraction for Liquid culture product of K346007(Ag43)

1. Used FastGene Plasmid Mini Kit(Nippon Genetics)
2. Elutioned in 50 ul
3. First we eluted in collection tube. then moved in micro-centrifuge tube.

Electrophoresis

Electrophoresis for plasmid extraction product(Ag43).

1. Prepared 1% Agalose gel and added EtBr then pre-migration for 30 min.
2. 1 ul 1kb ladder, 1.2 ul plasmid extraction product(1 ul is DNA solution and 0.2 ul is loading dye) added then migtrated.

Glycerol stock

Made glycerol stock of K346007 (Ag43).

1. Parts written above were incubated in LBC.
2. 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 plasmid extraction product)from our calculation. There are no insurance of succession of digestion.

• Ag43(Insert)

5190bp(Ag43 + pSB1C3) DW

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))

1. Added 5 ul of NaoAc , 1.5 ul of glycogen and 125 ul of 100% ethanol to 50 ul DNA solutions.
2. Centrifuged at 15000 rpm, 10min at 4C.
3. Removed supernatant and added 220 ul of 70% ethanol.
4. Centrifuged at 15000 rpm, 5 min at 4C.
5. Removed supernatant and air drying at 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.

1. Prepared 1% Agalose gel and added EtBr then pre-migration for 30 min.
2. Added 1kb ladder, Ligation product(1 ul) and digestion products (control:each solutions 1 ul).
3. Migtrated for 30 min.

Transformation

Transformation for K346007(Ag43)+B0015(dT) on pSB1AK3.

1. Added DNA soltions (Ligation products) 1 ul to DH5α compitent cell.
2. Incubated on ice for 30 min.
3. Added 600 ul of LB to transformed DH5α solution.
4. From 700 solution(100 ul is DH5α and 600 ul is LB), 100 ul add to 900 ul of LB(x10 solution)
5. Spread 300 ul from 600(700-100) ul and 1000 ul of LB&DH5α solution to each LBA plates.
6. Incubated for 19 hrs.