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Team:NYMU-Taipei/results/lab-journals
From 2011.igem.org
Contents |
7/1-7/2
- inoculate AMB-1 in (MSGM from frence)
- prepare the AMB-1 medium
7/3-7/9
- Separate AMB-1 culture
- Plasmid extraction of pRK415
- Stock AMB-1 at 80 。C DMSO
7/10-7/16
- Check the pRK475 extracted from AMB-1 on 7/8 with EcoR1
- Prepare MSGM wolfe’s solution
7/17-7/23
- Transformation the RFP,CFP,YFP,GFP,R-Luc,pUC19,pUC18 respectively into DH5 alpha.
- Plate the transformed competent cell on the LB+Amp plate.
- Prepare the DMEM
- Culture the J774 macrophage.
- Pick up the DH5 alpha colonies which are transformed on 7/18 that grow on the Amp plate,and inoculate them in the *LB+Amp.
- Transformation the pT7,RBS,tetR,GFP,ECFP,mcherry,RFP respectively into the DH5 alpha.
- Plamid extraction
- Inoculate the DH5 alpha in LB+Amp O/N.
- DH5 alpha transformed with RFP are streak out on the plate containing the KAN.
- Electroporation
- Recover the DH5 alpha and streak out them on the plate.
- Plasmid extraction to conform the result of electroporation.
- Use the microscope to exam the AMB-1 in the liquid culture.
- Prepare the AMB-1 medium.
- Stock the transformed DH5 alpha at -80 。C.
- Plasmid extraction.
- Inoculate the competent cells transformed with CFP,YFP,GFP,R-Luc,pUC19,pUC18 in LB+KAN;the competent cells *transformed with RFP in LB+Amp.
- Culture the J774cell cell lines in the DMEM.
- Prepare the kanamycin stock.
- Inoculate the AMB-1 in the new MSGM.
- Plasmid extraction.
- Stock DH5 alpha transformed with RFP,CFP,GFP, pUC19 at -80。C.
7/24-7/30
- Genomic DNA extraction of AMB-1.
- Cut the NotI site on the backbone that respectively contain pT7, RBS34 ,Tet-R, GFP and run gel to extract the band of *RBS vector, Tet-R and GFP.
- Ligate the Tet-R(vector) and the GFP(insert) ,then transform it to the competent cell
- Change the media of the J774 cell lines.
- Passaging cells.
- Examine the growth of AMB-1.
- RE cutting and run gel to extract the correct band of mcherry.
- Streak out the DH5 alpha which are transformed respectively with pT7,tetR+GFP on the plate.
- Plasmid extraction of pT7 and tetR+GFP.
- RE check the if tetR+GFP exist in the plasmid.
- Prepare the LB and autoclave it.
- Tet R+GFP Recheck
- Inoculate the pRKm415 in the LB+ KAN.
- Genomic DNA extraction of AMB-1.
- PCR the Pmsp3 segment.
- Stock pRKm415 at -80。C.
- Plasmid extraction of pRKm415.
- Restriction enzyme cutting :rbs34(S/P)and tetR+ GFP(RS+TR) (X/P)
- Run gel and extract the band of tetR+GFP and RBS34.
- Incubate the DH5 alpha with RBS34 at 37。C.
- RE cutting :tetR(S/P) and GFP(RS+TR) (X/P).
- Ligation of TetR(vector) and GFP(RS+TR)(insert)
- Transform tetR+ GFP(RS+TR) to competent cells.
- Stock the competent cell transformed with RBS34 at -80。C.
- Plasmid extraction from the competent cells that are transformed with RBS34.
- RE cutting:tetR(S/P) and GFP(RS+TR)(X/P)
- Incubate at 37。C for 90 min.
- Incubate the competent cells that are transformed with (tetR+GFP(RS+TR)).
- Ligation :tetR(vector) insert(GFP(RS+TR))
- Transform the tetR+GFP(RS+TR) into competent cell.
- Genomic DNA extraction of AMB-1.
- J774 cell lines passaging.
- Plasmid purification of tetR+GFP(RS+TR).
- RE check :tetR+GFP(RS+TR) at E/P site
- Ligation: tetR+GFP(RS+TR)
- Run gel and extract the tetR+GFP(RS+TR) band and ligate it with the RBS34.
- pT7-teto oligonucleotide annealing
- ligation:RBS34(vector)+pT7-teto(insert)
- transform pT7-tetO(E/S)+RBS34(E/X)
- incubate the competent cells that are transformed with tetR+GFP(RS+TR)
- ligation:RBS34(S/P)+tetR+GFP(RS+TR) (X/P)
- RE cutting:RBS34(S/P)
- Run gel to check if the length of tetR+ GFP(RS+TR) (X/P) band is correct.
- Ligation:RBS34(S/P)(vector)+tetR+GFP(RS+TR)(insert).
- Transformation the ligation product :BS34(S/P)(vector)+tetR+GFP(RS+TR)(insert) into the competent cells.
7/31-8/6
- Plasmid extraction of tetR+GFP(RS+TR),RBS+tetR+GFP(RS+TR),pT7-teto+RBS34
- RE check:pT7-teto+RBS34(Aatll&Pstl)
- Incubate the competent cells that are transformed with tet-R+GFP(RS+TR) on the plate.
- Amplify the segment of Pmsp13,mms13,pmms16,minC by PCR.
- plasmid extraction :Tet-R/GFP(RS+TR)
- RE cutting:tet-R(S/P),GFP(RS+TR)(X/P)
- Run gel and extract the correct band of tet-R(S/P) and GFP(RS+TR)(X/P).
- Amplify the minC segment by PCR.
- RE cutting:RBS34(E/X),Tet-R(X/P),GFP(RS+TR)(S/P)
- Passaging the J774 macrophage cell lines.
- Ligation: tet-R(S/P) +GFP(RS+TR)(X/P).
- Ligation: pT7-teto+RBS34
- Transformation
- pT7-teto+RBS34
- tet-R+GFP(RS+TR)(fridge)
- tet-R+GFP(RS+TR)(RE cutting)
- PCR product clean up :to purify the PCR product of minC for further annealing.
- Inoculate the competent cells which are transformed with pT7-teto+RBS34,
- tet-R+GFP(RS+TR)(fridge),tet-R+GFP(RS+TR)(RE cutting) respectively on the Amp added plate.
- Passaging the J774 macrophages.
- Plasmid extraction:pT7-teto+RBS34,tet-R+GFP(RS+TR)(fridge),tet-R+GFP(RS+TR)(RE cutting)
- Genomic DNA extraction of AMB-1.
- Amplify the mms13,pmms16,pmsp3 by PCR using the AMB-1 genomic DNA as template.
- RE check:TetR-GFP(RS+TR) (X/P)and pT7-teto+RBS34(Atall&Pstl)
- Ligation: TetR-GFP(RS+TR) (X/P)
- transformation
8/7-8/13
- Plasmid purification
- RE cutting:
- RBS34,tet-R+GFP(RS+TR)(fridge)(S/P)
- RE cutting(X/P)
- pT7(S/P)
- amplify the mms13,pmsp3,pmms16 fragments by PCR
- ligation” pT7”+”RBS34+tetR+GFP(RS+TR)“(fridge)
- ” pT7”+”RBS34+tetR+GFP(RS+TR)“ (RE)
- insert:vector=3:1
- PCR minC&invasion
- Run gel :check minC and invasin
- RE cutting->ligation
- minC(X/P)pT7-tetO+RBS34(S/P)->transform to DH5 alpha
- pT7(S/P) RBS34+tetR+GFP(X/P)->transform to BL21-DE3-pLys
- genomic DNA extraction from AMB-1.
- Ligation:pT7-teto-RBS(S/P)+minC(X/P)
- Incubate the transformed competent cells:
- pT7X2
- RBS34-tetR+GFP(RS+TR)(RE)X2
- RBS34-tetR-GFP(RS+TR)(fridge)X2
- Plasmid purification of
- RBS34-tetR+GFP(RS+TR)(RE)
- RBS34-tetR-GFP(RS+TR)(fridge)
- RE cutting
- pT7
- RBS34-tetR+GFP(RS+TR)(RE)
- RBS34-tetR-GFP(RS+TR)(fridge)
- Run gel->gel extraction
- 1)
- Ligation
- pT7(vector)(S/P)
- RBS34-tetR-GFP(RS+TR)(insert)(X/P)
- 2)
- Ligation
- pT7(vector)(S/P)
- RBS34-tetR-GFP(RS+TR)(insert)(X/P)
- Amplify the pmms16,pmsp3,mms13 fragment by PCR.
- Genomic DNA extraction of Yersinia genomic DNA.
- AMB-1 genomic DNA PCR with universal primer.
- Pmm16 PCR with pmms16 primer.
- tetR-GFP(RS+TR)->O
- RBS34->O
- RBS34+tetR-GFP->no parts
- pT7+tetO+RBS34->X
- pT7+tetO+RBS34+minC->no parts
- RE cutting E/P
- Check pT7 & pT7+tetO+RBS34
- RE cutting :
- minC (EP)
- tetR(EP)
- take the minC and tetR fragment to do the ligation
- Yersinia genomic DNA gel extraction
- PCR invasin
8/14-8/20
- Genomic DNA extraction of listeria monocytes.
- PCR the LLO sequence from it.
- PCR mms13
- Check parts
- transformation
- RBS34 (-80。C)
- tetR-GFP(RS+TR)fridge
- tetR-GFP(RS+TR)RE
- passaging the J774 cell lines.
- Change the medium.
- Incubate the competent cells:
- pT7-tetO-RBS34
- RBS34(-80。C)
- tetR+GFP(RS+TR)RE
- tetR+GFP(RS+TR)fridge
- passaging the J774 cell lines.
- plasmid purification:
- pT7-tetO-RBS34
- RBS34(-80。C)
- tetR+GFP(RS+TR)RE
- tetR+GFP(RS+TR)fridge
- RE cut
- pT7-tetO-RBS34(S/P)
- RBS34(-80。C)(X/P)
- tetR+GFP(RS+TR)RE(S/P)
- tetR+GFP(RS+TR)fridge(X/P)
- ligation
- RBS34(S/P):2ul
- tetR+GFP(X/P):12ul
- transformation
- RBS34-tetR-GFP(RS+TR)fridgeX2
- pT7 biobrickX2
- check
- Pmms16(small)KOD
- Pmms16(large)KOD
- minC KOD0819
- mms13 touch-d(2)
- mms13 touch-d(3)
- mms13 touch-d(4)
- minC PCR clean up
- annealing:
- trunk-rbs(Pmsp3)-R/F
- rbs(pmsp3)-R/F
- trunk-rbs(Pmsp3)F: trunk-rbs(Pmsp3)R=1:1
- rbs(Pmsp3)F: rbs(Pmsp3)R=1:1
- RE check:
- minC(AgeI)
- invasin(ClaI)
- PBS32(S/P)
- Plasmid purification
- pT7(clean)
- pT7(gel)
- RBS34-tetR-GFP(RS+TR)(clean)
- RBS34-tetR-GFP(RS+TR)(gel)
- RE check
- pT7(Xhol)
- RBS34-tetR-GFP(RS+TR)(E/P)
8/21-8/27
- Ligation
- pT7&RBS34-tetR-GFP(RS+TR)
- gel extraction LLO
- transformation
- pT7+RBS34-tetR-GFP into competent cells
- ligation
- RBS32(S/P)
- LLO(X/P)
- PCR check pT7+RBS34-tetR-GFP
- PCR invasin
- Transformation PBS-LLO
- Plasmid purification:
- RBS32+LLO1
- RBS32+LLO2
- pT7-RBS34-tetR-GFP2
- pT7-RBS34-tetR-GFP3
- pT7-RBS34-tetR-GFP4
- pT7-RBS34-tetR-GFP5
- incubate competent cells that are transformed with
- pT7-RBS34-tetR-GFP,RBS32-LLO
- colony PCR
- pT7-RBS34-tetR-GFP(RS+TR)
- PCR clean up “invasin”
- transformation
- pT7-RBS34-tetR-GFP(RS+TR)
- plasmid purification
- pT7 & RBS34-tetR-GFP(RS+TR)
- RE cutting
- pT7(vector)(S/P)
- RBS34-tetR-GFP(RS+TR)(insert)(X/P)
- Gel extraction
- RBS34-tetR-GFP(RS+TR)
- RE digest
- RBS34-tetR-GFP(RS+TR)(X/P)
- pT7(S/P)
- RBS32-LLO(1)(X/P)
- RBS32-LLO(2)(X/P)
- Invasion(X/P)
- pT7-RBS34-tetR-GFP(RS+TR)(E/P)
- RBS32(S/P)
- PCR clean up:pT7(S/P),invasion(X/P),RBS32(S/P)
- Gel extraction:RBS34-tetR-GFP(RS+TR),RBS32+LLO(X/P)
- Transformation to BL21(DE3)
- RBS34-tetR-GFP(RS+TR)
- Incubate competent cells that contains RBS32.
- Ligation
- “pT7”+"RBS+LLO“
- “RBS32”+”invasion”
- Plasmid purification from RBS32 broth,plate.
- RE cutting
- RBS32-LLO(S/P)
- ECFP(X/P)
- LLO check
- Pick up the single colonies from the competent cells that are transformed with
- “pT7+RBS+LLO“
- “RBS32+invasion”
- Stock BL21 that are transformed with
- pT7-RBS34-tetR-GFP(RS+TR)
- measure the GFP (BL21)strengh
- pT7-RBS34-tetR-GFP(RS+TR)-IPTG induction
- plasmid purification of pT7-RBS34-tetR-GFP(RS+TR) (BL21)
- plasmid purification of“pT7+RBS+LLO“
- plasmid purification of“RBS32+invasion”
- PCR minC,invasion ,LLO
- RE cutting invasion (X/P)
- RBS34(X/P)
- Ligation
- 1
- Invasion(X/P)
- RBS(X/P)
- 2
- LLO(X/P)
- RBS34(X/P)
- Transformation
- Invasion into DH5 alpha
- LLO into DH5 alpha
- Plasmid purification of
- “pT7+RBS+LLO“
- “RBS32+invasion”
- PCR clean up:minC
- PCR
- MinC,invasion,LLO
- Plasmid purification:LLO invasin
- RE check:
- LLO(E/P)
- Invasin(E/P)
- RE cutting
- RBS34(X/S)
- PCR clean up PBS34
- minC
- LLO
8/28-9/3
- RE check plasmid “invasion”&”LLO”
- Invasin(ClaI)
- LLO(ClaI)
- IPTG-induction
- Pick up single colonies of cells that respectively contain Invasin,LLO,minC ,then incubate it in the LB.
- Plasmid purification from the competent cells that contain Invasin ,LLO,minC.
- RE check invasin(E/S)
- LLO (E/P)
- RE cutting minC
- (E/S)
- Genomic DNA extraction from AMB-1.
- PCR LLO and Invasin
- Run gel and extract the correct band of LLO.
- RE cutting
- LLO(X/P)
- tetR+GFP(S/P)
- tetR(S/P)
- GFP(S/P)
- LLO clean up
- Check invasion
- RE cutting->ligation
- Vector tetR(X/P)backbone
- Insert LLO(X/P)
- PCR Invasin
9/4-9/10
- Plasmid purification of pT7,ECFP,RBS32+LLO,RBS32+invasin
- RE check
- pT7 (Xhol)
- ECFP(E/S)
- RBS32+LLO(E/P)
- RBS32+invasion(E/P)
- Passaging the J774 cell lines.
- Incubate the competent cells with araC,pT7 respectively.
- Plasmid purification of
- pT7 09-1
- pT7 09-2
- pT7 10-3
- pT7 10-4
- Arac1
- Arac2
- RE check
- pT7 09-1(P)
- pT7 09-2(P)
- pT7 10-3(P)
- pT7 10-4(P)
- Arac1(E/P)
- Arac2(E/P)
- Plasmid purification
- K12-MG655 genomic DNA
- RE cutting
- YFP(E/P)
- pUC19(E/P)
9/11-9/28
- minC nested PCR product->minC with mutated prevous PstI site.->prepared for ligation pUC19.
- minC check(E/P)
- RE cutting
- pUC19(E/P),pUC19(E/P)-> PCR Clean up
- ligate minC+pUC19
- transform to DH5 alpha
- pUC57
- pUC19
- biobrickB13
- inoculate the transformed DH5 alpha in LB.
- plasmid purification
- RE check
- minC(AgeI)
- transformation(LLO invasion ori+p+rep pUC19)
- plasmid purification
- ligation mms13
- RE check
- ori&PmspI+invasine
- ori&PmspI+LLO
- ori&PmspI+rep
- RE cutting
- pUC19+ori&PmspI+rep (E/S)
- pUC19+ori&PmspI+rep(E/S)
- incubate the competent cells that are transformed with pUC19+ori&Pmsp+rep
- plasmid purification
- transformation
- pYMB(RBS)
- pYMB(RBS-trunc)
- pYMB(RBS-GFP)
- pYMB(RBS-trunc-GFP)
- into DH5 alpha
- colony PCR
- prepare EMSGM +Amp plate
- transform the rbs-GFP0pUC19 into the competent cell.
- RE cutting
- pSB1C3(X/P)
- RE cutting
- Pmsp1+RBS(S/P)
- minC&invasin &LLO(X/P)
