Team:NYMU-Taipei/results/lab-journals

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 +
 
 +
==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)

Latest revision as of 20:00, 5 October 2011

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Contents

7/1-7/2

7/3-7/9

7/10-7/16

7/17-7/23

7/24-7/30

7/31-8/6

8/7-8/13

8/14-8/20

8/21-8/27

8/28-9/3

9/4-9/10

9/11-9/28

Retrieved from "http://2011.igem.org/Team:NYMU-Taipei/results/lab-journals"