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From 2012.igem.org

• 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

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• 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.

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• 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).

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• 7th

Liquid culture

Liquid culture in LBC(Ag43).

1. Picked up two colonies from each plates.
2. Both of colonies were dipped in 2ml LBC, and then we cultivate them in 38℃.
   

However, one of them cultivated only 8 hours. It's for glycerol stock.

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

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

All DNA solutions were digested. 3A assembly protocol required Ligation reaction should be in total 25ul solution.

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

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!!!!

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• 8th

• (pT7 + RBS)

Transformation

Transformation for pT7+RBS+pSB1K3

1. Added DNA soltions (Ligation products) 1ul to DH5α compitent cell.
2. Stood on ice in 30min.
3. Added 600ul of LB to transformed DH5α solution.
4. Pre-cultivate in 2hrs
5. Splead 300ul of LB&DH5α solution to LBK.
6. Cultivated

• K346007(Ag43)

mini-prep

mini-prep for Liquid culture product of K346007(Ag43)

1. Used FastGene Plasmid Mini Kit(Nippon Genetics)
2. Elutioned in 50ul
3. First we eluted in colection tube. then moved in Eppendorf tube.

Erectrophoresis

Erectrophoresis for mini-prep product(Ag43).

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

mini-prep result (With ligation result of pT7+RBS+pSB1K3)

Glycerol stock

Made glycerol stock of K346007 (Ag43).

1. Parts written above were cultivated 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 DNA mini-prep product)from our calculation. There are no insurance of succession of digestion.

• Ag43(Insert)

5190bp(Ag43 + pSB1C3)

DW

DNA solution	48ul
EcoRI	1ul
SpeI	1ul
10xH buffer	6ul
4ul
――――――――――――
Total	60ul

• dT(Vector)

3318bp(Ag43 + pSB1AK3)

DNA solution	8ul
EcoRI	1ul
XbaI	1ul
10xM buffer	2ul
DW	8ul
――――――――――――
Total	20ul

Digestion result image

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 5ul of NaoAc , 1.5ul of glycogen and 125ul of 100% ethanol to 50ul DNA solutions.
2. Centrifuged in 15000rpm, 10min at 4C.
3. Remove supernatant and added 220ul of 70% ethanol.
4. Centrifuged in 15000rpm, 5min at 4C.
5. Remove supernatant and air drying in room temperature then added 10ul of DW.

Ligation

All DNA solutions were digested. Used Ligation Mighty Mix(TakaraBio)

Ligation Mighty Mix	5ul
Insert: Ag43	2ul
Vector: dT	2ul
DW	1ul
――――――――――
Total	10ul

Ligation reaction recipe was written below.

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 in 30min.
2. Added 1kb ladder, Ligation product(1ul) and digestion products (control:each solutions 1ul).
3. Migtrated in 30min.

Electrophoresis results

Transformation

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

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

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• 9th

pT7 + RBS (3A Assembly) and Ag43 + dT (standard assembly) ligation products were transformed and cultivated then some colonies were formed (12~16 colonies) so we confirmed really insert DNA (3A:pT7 and RBS, standard:Ag43 and dT) were inserted to vector or not by colony PCR.

Colony PCR

Colony PCR for assembly products.Each product reacted recipes written below.

1. picked up each 12(16 is best but there were only 12 colonies) colonies from LB plates by Autoclaved toothpicks.
2. Dipped into 10ul DW in 1.5ml eppendorf tubes.
3. from 10ul DW, 4ul was added in PCR reaction solution (below), 6ul was mixed with 200ul LB.
4. Ran PCR machine in recipe below.
5. Electrophoresis for confirmation of PCR results.

PCR reaction solution

DNA solution	4ul
KapaTaq ready mix	5ul
BioBrick prefix forward primer	0.5ul
BioBrick suffix reverse primer	0.5ul
――――――――――――――――――――――――――
Total	10ul

PCR recipe

(pT7 + RBS)

Number	Degree	Second
1	94	120
2	94	30
3	68	60
4	4	HOLD

Cycle:2~3 x 40

(Ag43 + dT)

Ag43 + dT (+ Biobrick prefis & suffix) is about 3290bp. Extension step needed >180seconds.

Number	Degree	Second
1	94	120
2	94	30
3	68	180
4	4	HOLD

Cycle:2~3 x 35

Electrophoresis results

Electrophoresis for (pT7 + RBS) in 2% agarose gel and (Ag43 + dT) in 1% agarose gel.

pT7 + RBS on pSB1K3 bbp-Insert-bbs:86bp

Ag43 + dT on pSB1AK3 bbp-Insert-bbs:3290bp

We couldn't confirm insert DNA were really ligated with Vector or not. Next we tried confirmation of insert DNA by Electrophoresis of mini-prep products. For mini-prep, we needed do liquid culture.

Liquid culturing

Liquid culture for mini-prep((pT7 + RBS) on pSB1K3 and (Ag43 + dT) on pSB1AK3).

1. Prepared 1800ul LB solutions.
2. To these LB solutions, added 200ul of LB solutions (colony PCR solutions were pre-cultivated in about 2hrs) and each antibiotics(Km (for (pT7 + RBS) on pSB1K3) and Amp(for (Ag43 + dT) on pSB1AK3)).
3. Cultivated 15hrs30min.

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• 10th

Mini-prep

Mini-prep for some colonies (we selected colonies which showed more correct bands than other colonies, pT7+RBS were No.4, 5, 9, 10 colonies and Ag43 + dT were No.1, 2, 3, 4 colonies)cultivated in LBA(Ag43 + dT) and LBK(pT7 + RBS) in 15hrs 30min.

1. Mini-prep for LB culturing products along the protocol of FastGene Plasmid Mini kit(Nippon Genetics).
2. Electrophoresis (Used pre-migrated 1% agarose gels with 5ul EtBr)in 30min.

Electrophoresis resulsts

pT7 + RBS on pSB1K3(Total 2247bp)

Ag43 + dT on pSB1AK3(Total 6444bp)

To confirm more about insert DNA, we tried to digest these DNA with EcoRI and PstI.

Digestion

Digestion for confirmation of insert DNA. Each DNA mini-prep products were digested with E & P.

Digestion recipe pT7-RBS

pT7-RBS	1,5ul
EcoRI	1ul
PstI	1ul
10xH buffer	2ul
DW	14.5ul
――――――――――――
Total	20ul

Digestion recipe Ag43-dT

Ag43-dT	4ul
EcoRI	1ul
PstI	1ul
10xH buffer	2ul
DW	12ul
――――――――――――
Total	20ul

Digestioned at 37c in 2hrs.

Digestion results

pT7+RBS

Ag43+dT

Insert DNA were correct we thought. We tried digestion for 3A Assembly[pT7-RBS + Ag43-dT + pSB1C3]

Digestion

Digestion for 3A Assembly.

pT7-RBS

DNA	17ul
EcoRI	1ul
SpeI	1ul
10xH buffer	3ul
DW	8ul
――――――――――――
Total	30ul

Ag43-dT

DNA	12.5ul
XbaI	1ul
PstI	1ul
10xH buffer	2ul
DW	3.5ul
――――――――――――
Total	20ul

pSB1C3

DNA	20ul
EcoRI	1ul
PstI	1ul
10xH buffer	3ul
DW	5ul
Total	30ul

Reacted in 2hrs at 37c.