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{{:Team:NYMU-Taipei/Header}} ='''Our Goal'''= =='''GFP'''== '''Purpose''' Insert lva tag to the GFP by using PCR and ligate to pLac. '''Material''' [http://partsregistry.org/Part:BBa_R0010 BBa_R0010]''':''' pLac [http://partsregistry.org/Part:BBa_E0240 BBa_E0240]''':''' RBS([http://partsregistry.org/Part:BBa_B0034 BBa_B0034]) +GFP([http://partsregistry.org/Part:BBa_E0040 BBa_E0040]) +Terminator([http://partsregistry.org/Part:BBa_B0015 BBa_B0015]) '''Parts Length''' {| border="5" |colspan=3 align="center"|'''DNA Length''' |- |Part||Part Length||Length in pSB1A2 |- |E0240||876bp||1114bp |- |E0240+lva||909bp||1147bp |- |R0010+E0240+lva||1117bp||1355bp |} =='''RFP'''== '''Purpose''' Insert lva tag to the RFP by using PCR and ligate to pLac. '''Material''' BBa_R0010''':'''pLac [http://partsregistry.org/Part:BBa_I13507 BBa_I13507]''':''' RBS(BBa_B0034)+RFP([http://partsregistry.org/Part:BBa_E1010 BBa_E1010])+Terminator(BBa_B0015) '''Parts Length''' {| border="5" |colspan=3 align="center"|'''DNA Length''' |- |Part||Part Length||Length in pSB1A2 |- |I13507||861bp||1099bp |- |I13507+lva||894bp||1132bp |- |R0010+I13507+lva||1102bp||1340bp |} =='''CFP'''== '''Purpose''' After ligate CFPlva to Terminator and RBS , ligate to pLac. '''Material''' BBa_R0010''':''' pLac BBa_B0034''':''' RBS [http://partsregistry.org/Part:BBa_E0022 BBa_E0022]''':''' CFPlva BBa_B0015''':''' Terminator '''Parts Length''' {| border="5" |colspan=3 align="center"|'''DNA Length''' |- |Part||Part Length||Length in pSB1A2 |- |E0022||762bp||1000bp |- |E0022+B0015||899bp||1137bp |- |B0034+E0022+B0015||919bp||1157bp |- |R0010+B0034+E0022+B0015||1127bp||1365bp |} =='''YFP'''== '''Purpose''' After ligate YFPlva to Terminator and RBS , ligate to pLac. '''Material''' BBa_R0010''':''' pLac BBa_B0034''':''' RBS [http://partsregistry.org/Part:BBa_E0032 BBa_E0032]''':''' YFPlva BBa_B0015''':''' Terminator '''Parts Length''' {| border="5" |colspan=3 align="center"|'''DNA Length''' |- |Part||Part Length||Length in Plasmid |- |E0032||762bp||1000bp |- |E0032+B0015||899bp||1137bp |- |B0034+E0032+B0015||919bp||1157bp |- |R0010+B0034+E0032+B0015||1127bp||1365bp |} ='''Our Experiment Data '''= == 2010-07-22 == 1. Extract B0034(RBS) from 2010 plate 1 2U Extract B0015(Terminator) from 2010 plate 1 23L 2.Transform into 15ul competent cells. 3.Add 85ul LB after heat shock. 4.Culture for 30 minutes. 5.80ul for transformation ; 20ul for liquid culture 6.Incubate at 6:00pm. == 2010-07-23 == 1.Extract RBS and Term(abbreviation for terminator) cultured in 7/22. 2.Measured the nano drop. B0015:26.94ng/ul B0034:45.59ng/ul 3.Digest B0034(SP) and B0015(XP) at 11:30am 4.Run the gel for B0015. {{:Team:NYMU-Taipei/GEL|NYMU G0723.jpg|-|c= 2% agarose, 90V, 40min {{:Team:NYMU-Taipei/GELC|= |0: -|-|-|n|= |1: -|-|-|n|= |2: -|-|-|n|= |3: -|-|-|n|= |4: Marker 100bp|-|-|n|= |5: Term|129bp|none|f|= |6: Term|129bp|none|f|= |7: |||n|= |8: |||n|=}} }} 5.Measured the nano drop. B0034:3.85ng/ul 6.Purify B0034. 7.Ligate YFPlva and CFPlva to Term. 8.Transform into 30ul competent cells. 9.PCR to make GFPlva and RFPlva front parts. {{:Team:NYMU-Taipei/GEL|NYMU G07.jpg|{{:Team:NYMU-Taipei/PCR|2m|30s|30s|90s|10m|rp=VR|fp=VF2|polName=pfu|pol=0.5|ddH20=39.5|cycles=35}}|c= 2% agarose, 90V, 40min {{:Team:NYMU-Taipei/GELC|= |0: -|-|-|n|= |1: Marker 100bp|-|-|n|= |2: -|-|-|n|= |3: GFP front part|||n|= |4: RFP front part|||n|= |5: |||none|n|= |6: |||none|n|= |7: |||n|= |8: |||n|= }} }} ==2010-07-24== 1.Do 3 in 1 and PCR to check CFPlva+Terminator ,YFPlva+Terminator ,RBS , Terminator. 2.Run the gel. {{:Team:NYMU-Taipei/GEL|NYMU G0724 1.jpg|{{:Team:NYMU-Taipei/PCR|2m|15s|20s|90s|5m|rp=VR|fp=VF2}}|c= 2% agarose, 90V, 40min {{:Team:NYMU-Taipei/GELC|= |1: Marker 1kb|-|-|n|= |2: positive|-|-|n|= |3: negative|-|-|n|= |4: CFPlva+Terminator|||n|= |5: CFPlva+Terminator|||n|= |6: CFPlva+Terminator|724|none|f|= |7: CFPlva+Terminator|758|none|f|= |8: YFPlva+Terminator|-|-|n|= |9: YFPlva+Terminator|-|-|n|= |10: RBS|-|-|n|= |11: Terminator|-|-|n|= |12: Marker 100bp|-|-|n|= }}positive control:GFP template}} 3.PCR to make the whole parts GFPlva and RFPlva. 4.Run the gel. {{:Team:NYMU-Taipei/GEL|NYMU G0724 2.jpg|{{:Team:NYMU-Taipei/PCR|2m|15s|20s|90s|5m|rp=VR|fp=VF2}}|c= 2% agarose, 90V, 40min {{:Team:NYMU-Taipei/GELC|= |1: Marker 100bp|-|-|n|= |2: positive|-|-|n|= |3: GFPlva whole part|||m|= |4: RGPlva whole part|||m|= |5: negative|-|-|n|= |6: Marker 100bp|724|none|n|= }}positive control:GFP template}} 5.Digest GFPlva(XP) and RFPlva(XP). 6.Measure the nano drop. GFPlva:8.66ng/ul RFPlva:7.35ng/ul 7.Ligate GFPlva and RFPlva to psB1A2. 8.Tranform into 30ul competent cells. ==2010-07-26== 1.Digest RBS(SP), CFP(XP), YFP(XP) and Term(P) 2.Run the gel for . 3.Digest CFPlva and YFPlva. 4.Ligate CFPlva and YFPlva to Term. ==2010-07-28== 1.Do 3 in 1 to check GFPlva ,RFPlva , YFPlva+Term and CFPlva+Term. 2.Run the gel. {{:Team:NYMU-Taipei/GEL|PICTURE|{{:Team:NYMU-Taipei/PCR|2m|15s|20s|90s|5m|rp=VR|fp=VF2}}|c= 2% agarose, 90V, 40min {{:Team:NYMU-Taipei/GELC|= |1: Marker 100bp|-|-|n|= |2: positive|-|-|n|= |3: negative|-|-|n|= |4: YFPlva+Terminator|1137bp|1000bp|f|= |5: CFPlva+Terminator|1137bp|1000bp|f|= |6: RFPlva|1147bp|238bp|m|= |7: GFPlva|1132bp|238bp|m|= |8: Marker 100bp|-|-|n|= }}positive control:GFP template}} ==2010-07-29== 1.Extract GFPlva and RFPlva cultured on 7/29. 2.Digest GFPlva(X,NdeI) and RFPlva(X,NcoI) for checking if they were correct. Digest YFPlva(E) ,CFPlva(E) and Term(E) for ligation. 3.After 10 minutes ,degrade EcoRI by rising the temperature to 80 for 5 minutes. 4.Run the gel for YFPlva ,CFPlva and Term and purify them. 5.Digest YFPlva(ES) ,CFPlva(ES) and Term(EX). 6.Measure the nano drop. 7.Run the gel for YFPlva(ES) ,CFPlva(ES) ,Term(EX) ,GFPlva and RFPlva. 8.Ligate CFPlva(ES) and YFPlva(ES) to Term(EX). 9.PCR the whole part for GFPlva and RFPlva again. ==2010-07-31== 1.Do 3 in 1 for YFPlva+Term and CFPlva+Term. 2.Run the gel for YFPlva+Term ,CFPlva+Term ,RFPlva and GFPlva. 3.Ligate YFPlva(ES) to Term(EX). 4.PCR the front part of GFPlva and RFPlva for checking the condition is right or wrong. 5.Run the gel to make the front part of GFPlva and RFPlva. ==2010-08-02== 1.Do 3 in 1 for YFPlva+Term. 2.Digaest CFPlva+Term ,RBS(SP) ,Term(ES) and Term(E). 3.Measure the nano drop. 4.PCR to make the front part of GFPlva and RFPlva. 5.Ligate RBS(SP) to CFPlva+Term (XP). 6.Liquid culture for RBS. ==2010-08-04== 1.PCR to make the whole part of GFPlva and RFPlva. 2.Extract GFPlva ,RFPlva ,YFPlva+Term ,RBS. ==2010-08-05== 1.PCR for RFPlva, GFP without RBS, RFP without RBS, RBS+YFP+term, GFPlva. 2.Run gel for RBS+YFP+term, GFPlva, RFPlva, GFP without RBS, RFP without RBS [[Image:3159.jpg]] ==2010-08-06== 1.PCR for front part of GFP without RBS and front part of RFP without RBS 2.Extraction for YFP, GFPlva, RFPlva 3.Digestion 4.Run gel for RBS+YFP+term 5.PCR again for GFP without RBS, RFP without RBS 6.Purification for YFP, GFPlva, RFPlva 7.Nanodrop GFPlva(XP) 3.89ng/uL RFPlva(XP) 4.48ng/uL RBS+YFP+term 3.18ng/uL 8.Ligate CFP+term with RBS 9.Transformation ==2010-08-07== 1.Colony PCR for RBS+CFP+Term, GFP, RFP 2.Run gel for RBS+CFP+term, GFP, RFP ==2010-08-09== 1.Extraction for GFP, RFP 2.Colony PCR for GFP ==2010-08-10== 1.Digestion for GFPlva(XP), YFPlva(XP), RFPlva(XP), CFP+Term(XP) 2.Run gel for GFPlva(XP), YFPlva(XP), RFPlva(XP), CFP+Term(XP) ==2010-08-11== 1.Extract CFP+term 2.Real PCR for GFP wihout RBS, RFP without RBS 3.Ligate RBS with CPF+Term 4.Transform RBS with CPF+Term, pLac(R0010) ==2010-08-12== 1.Colony PCR for RBS+CFP+Term, pLac 2.Run gel for GFP without RBS, RFP without RBS, pLac ==2010-08-14== 1.Extract pLac 2.Digest pLac(SP) 3.Nanodrop pLac1 5.73ng/uL pLac2 2.70ng/uL pLac3 4.08ng/uL ==2010-08-17== 1.Ligate RBS with CFP+term 2.Transform RBS with CFP+term 3.Digest GFPlva(XP), RBS+YFPlva+term(XP) 4.Run gel for GFPlva(XP), RBS+YFPlva+term(XP) 5.Purify GFPlva(XP), RBS+YFPlva+term(XP) 6.Nanodrop GFPlva(XP) 8.87 ng/uL RBS+YFPlva+term(XP) 5.01 ng/uL 7.Culture GFPlva, RBS+YFPlva+term ==2010-08-18== 1.Extract GFPlva, RBS+YFPlva+term 2.Digest RBS, CFP+term 3.Ligate CFP+term with RBS ==2010-08-19== 1.Colony PCR for RBS+CFPlva+Term 2.Nanodrop RBS(SP) 21.38ng/uL CFP+Term(XP) 10.38ng/uL 3.Run gel for RBS+CFPlva+Term 4.Ligate RBS with CFPlva+term 5.Transform ==2010-08-20== 1.Colony PCR for RBS+CFPlva+term 2.Run gel for RBS+CFPlva+term ==2010-08-21== 1.Extract for RFP+CFPlva+term 2.Digest RBS+CFPlva+term(XP), RBS(SP) 3.Run gel for RBS+CFPlva+term 4.Purify RBS+CFPlva+term(XP), RBS(SP) 5.Nanodrop RBS1 2.96 ng/uL RBS2 2.02 ng/uL RBS+CFPlva+term 8.98 ng/uL ==2010-08-23== 1.Ligate pLac with RBS+GFPlva+term, pLac with RBS+YFPlva+term, pLac with RBS+RFPlva+term 2.Transform pLac with RBS+GFPlva+term, pLac with RBS+YFPlva+term, pLac with RBS+RFPlva+term ==2010-08-24== 1.Colony PCR for pLac+RBS+GFPlva+term, pLac+RBS+YFPlva+term, pLac+RBS+RFPlva+term ==2010-09-01== 1.Colony PCR for pLac+RBS+GFPlva+term 2.Extract RBS+CFPlva+term 3.Digest RBS+CFPlva+term(XP) 4.Run the gel for pLac+RBS+GFPlva+term,RBS+CFPlva+term(XP) 5.Ligate pLac with RBS+GFPlva+term, pLac wtih RBS+RFPlva+term, pLac with RBS+YFPlva+term, pLac with RBS+CFPlva+term ==2010-09-02== 1.Colony PCR for pLac+RBS+GFPlva+term, pLac+RBS+RFPlva+term, pLac+RBS+YFPlva+term, pLac+RBS+CFPlva+term 2.Run gel for pLac+RBS+GFPlva+term, pLac+RBS+RFPlva+term, pLac+RBS+YFPlva+term, pLac+RBS+CFPlva+term ==2010-09-04== 1.Extract pLac+RBS+GFPlva+term, pLac+RBS+RFPlva+term, pLac+RBS+YFPlva+term, pLac+RBS+CFPlva+term 2.Digest pLac+RBS+GFPlva+term(XP), pLac+RBS+RFPlva+term(XP), pLac+RBS+YFPlva+term(XP), pLac+RBS+CFPlva+term(XP) 3.Run gel for pLac+RBS+GFPlva+term(XP), pLac+RBS+RFPlva+term(XP), pLac+RBS+YFPlva+term(XP), pLac+RBS+CFPlva+term(XP) 4.Purify pLac+RBS+GFPlva+term(XP), pLac+RBS+RFPlva+term(XP), pLac+RBS+YFPlva+term(XP), pLac+RBS+CFPlva+term(XP) 5.Nanodrop pLac+RBS+GFPlva+term(XP)1 22.31 ng/uL pLac+RBS+GFPlva+term(XP)2 14.16 ng/uL pLac+RBS+RFPlva+term(XP) 8.6 ng/uL pLac+RBS+YFPlva+term(XP) 13.52 ng/uL pLac+RBS+CFPlva+term(XP)1 21.79 ng/uL pLac+RBS+CFPlva+term(XP)2 7.87 ng/uL ==2010-09-11== 1.PCR to make the front part and back part of the YFP and CFP. (in order to remove lva tag) 2.Run the gel for the front part and back part of YFP and CFP.(load 5ul) 3.While running the gel ,we purified them. 4.After the gel result appeared ,run the gel for YFP and CFP which had been purified to extract the correct length.(load 20ul) {{:Team:NYMU-Taipei/GEL||{{:Team:NYMU-Taipei/PCR|2m|30s|30s|90s|10m|rp|fp|polName=pfu|pol=0.5|ddH20=39.5|cycles=35}}||c= 2% agarose, 90V, 40min {{:Team:NYMU-Taipei/GELC|= |1: positive|-|-|n|= |2: Marker 100bp|-|-|n|= |3: YFP front part|1099|-|n|= |4: YFP back part|241||n|= |5: CFP front part|1099||n|= |6: CFP back part|241||n|= |7: negative|-|-|-|n|= |8: |||n|= }} }} {|border="1" |- |||template||forward primer||reverse primer |- |positive||RFP||VF2||VR |- |YFP front part||pLac+RBS+YFPlva+Term||VF2||XFP lva remover rp |- |YFP back part||pLac+RBS+YFPlva+Term||XFP lva remover fp||VR |- |CFP front part||pLac+RBS+CFPlva+Term||VF2||XFP lva remover rp |- |CFP back part||pLac+RBS+CFPlva+Term||XFP lva remover fp||VR |} Extract the gel and purify them. Measure the nano drop. YFP front part: YFP back part: CFP front part: CFP back part: Extract pLac+GFPlva ,pLac+RBS+YFPlva+Term and pLac+RBS+CFPlva+Term. Measure the nano drop. pLac+GFPlva: 170.51ng/ul pLac+RBS+YFPlva+Term: 276.08ng/ul pLac+RBS+YFPlva+Term: 287.46ng/ul pLac+RBS+CFPlva+Term: 116.21ng/ul pLac+RBS+CFPlva+Term: 252.12ng/ul ==2010-09-18== 1.Extract RFP. 2.Do the PCR to make the front part and back part of the RFPlva and to make the whole part of pLac+RBS+YFP+Term and pLac+RBS+YFP+Term. ==2010-09-30== 1.Run the gel for sspB, the front part of RBS+CFP+term, the front part of RBS+YFP+term, the front part of RBS+RFPlva+term, the back part of RBS+CFP+term, the back part of RBS+YFP+term, the back part of RFPlva and RFP 2.Purify sspB, the front part of RBS+CFP+term, the front part of RBS+YFP+term, the front part of RBS+RFPlva+term, the back part of RBS+CFP+term, the back part of RBS+YFP+term and RFP 3.Measure nanodrop sspB 34.73ng/uL the front part of RBS+CFP+term 15.82ng/uL the front part of RBS+YFP+term 35.53ng/uL the front part of RBS+RFPlva+term 76.10ng/uL the back part of RBS+CFP+term 34.89ng/uL the back part of RBS+YFP+term 36.27ng/uL RFP 79.82ng/uL == 2010-10-1== 1.PCR to make back part of RFPlva 2.Digest pLac(SP), sspB(PCR) and RFP(PCR) == 2010-10-4== 1.3-in-1 for pLac+RFP 2.Run the gel for pLac+RFP == 2010-10-7== 1.Extract RFPlva(PCR) RFPlva 100ng/uL 2.Digest RFPlva(XP) == 2010-10-9== 1.3-in-1 for K145205 2.PCR to make the whole part of pLac+RBS+YFP+term and pLac+RBS+CFP+term 3.Purify RFP(PCR)(XP) Nanodrop: RFP 42.3ng/uL 4.Ligate pLac(SP)+RBS+RFP+term(PCR)(XP) 5.Run gel for checking K145205 6.Purify pLac+BRS+RFP+term and pSB1C3+RFP Nanodrop: pLac+RFP1 72.8ng/uL pLac+RFP2 22.5ng/uL pSB1C3+RFP1 105.4ng/uL pSB1C3+RFP2 91.5ng/uL pSB1C3+RFP3 65.3ng/uL 7.Run gel for pLac+RBS+YFP+term and pLac+RBS+CFP+term(PCR) 8.Digest pLac+BRS+RFP+term(XP)and pSB1C3+RFP(XP) 9.Extract pLac+BRS+RFP+term(XP)and pSB1C3+RFP(XP) and purify them == 2010-10-11== 1.Digest pLac+RBS+CFP+term(XP) and pLac+RBS+YFP+term(XP) 2.Ligate pLac+RFPlva == 2010-10-12== 1.3-in-1 for pLac+RFPlva 2.Run the gel for checking pLac+RFPlva 3.Run the Gel for pLac+RBS+YFP+Term and pLac+RBS+YFP+Term(PCR)(digestion(XP)) 4.Run the gel for pLac+RFP(XP)and RFP+pSB1C3(XP) 5.Extract GFP, K145205 and pLac 6.Digest GFP(XP) and pLac(SP) 7.Purify pSB1A2(XP), pSB1C3(XP), pLac+RBS+RFP+term(XP), pLac+RBS+YFP+term(XP)and pLac+RBS+CFP+term(XP) == 2010-10-14== 1.Ligation for pLac+GFPlva, pLac+CFPlva, pLac+RFPlva, pLac+RBS+YFP+term with pSB1A2 == 2010-10-15== 1.Colony PCR for pLac+RBS+YFP+term, pLac+RBS+CFP+term 2.Run gel for pLac+RBS+YFP+term, pLac+RBS+CFP+term == 2010-10-21== 1.Colony PCR for pLac+RBS+GFP+term, pLac+RBS+YFP+term, pLac+RBS+CFP+term 2.Run gel for pLac+RBS+GFP+term, pLac+RBS+YFP+term, pLac+RBS+CFP+term {{:Team:NYMU-Taipei/Footer}}

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