Team:HokkaidoU Japan/Notebook/aggregation Week 1

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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

  1. Mixed 1 ul DNA solution with DH5α competent cells and incubated on ice in 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.


July 5th

Transformation

K346007(Ag43) was failed to cultivate on LBC plate. Transformation of K346007(Ag43) in DH5α.

  1. Add DNA solution(1 ul) into DH5α comeptent cell and stand on ice in 30 min.
  2. Pre-cultivated in 2 hrs.
  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 14 hrs and 30 mins
BBa_K542009 was Ag43 only part! And the part didn't have Biobrick suffix.


July 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 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.
  3. 16 hrs Cultivation.

Single colony isolation

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


July 7th

Liquid culture

Liquid culture in LBC(Ag43).

  1. Resuspended two colonies from each plates.
  2. Both of colonies were dipped in 2 ml LBC, and then we cultivate them in 38C.
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. We used FastGene Plasmid Mini Kit(Nippon Gene)
  2. Got 50 ul of DNA solutions.

Glycerol stock

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) 1 ml in 16 hrs 30 min.
  2. Add glycerol and Freeze at -80C

Electrophoresis

Erectrophoresis result

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

  1. Used 1% agarose gel.
  2. Added EtBr into TBE buffer and started Pre-migration.
  3. Migrated 1.2 ul of DNA solutions (1 ul is mini-prep products and 0.2 ul is Loading Dye) in 35 min.
  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 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.

  1. Added 3 ul of NaoAc, 1.5 ul of glycogen and 75 ul of 100% ethanol.
  2. Centrifuged in 14000 rpm, 30 min at 4C.
  3. Remove supernatant and added 220ul of 70% ethanol.
  4. Centrifuged in 15000 rpm, 15 min at 4C.
  5. 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

  1. Added DNA soltions (Ligation products) 1 ul to DH5α compitent cell.
  2. Stood on ice in 30 min.
  3. Added 600 ul of LB to transformed DH5α solution.
  4. Pre-cultivate in 2 hrs
  5. Splead 300 ul 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 50 ul
  3. First we eluted in colection tube. then moved in microcentrifuge tube.

Erectrophoresis

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

Erectrophoresis for mini-prep product(Ag43).

  1. Prepared 1% Agalose gel and added EtBr then pre-migration in 30 min.
  2. 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).

  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 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
Digestion result

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 in 15000 rpm, 10min at 4C.
  3. Remove supernatant and added 220 ul of 70% ethanol.
  4. Centrifuged in 15000 rpm, 5 min at 4C.
  5. 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

Erectrophoresis result

Confirmation of succession of ligation.

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

Transformation

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

  1. Added DNA soltions (Ligation products) 1 ul to DH5α compitent cell.
  2. Stood on ice in 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. Cultivated.