SsrA Experiment

From 2010.igem.org

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2% agarose, 90V, 40min
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|4: YFPlva+Terminator|1137bp|1000bp|f|=
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==2010-07-29==
==2010-07-29==

Revision as of 12:01, 27 October 2010


Contents

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

DNA Length
PartPart LengthLength in pSB1A2
E0240876bp1114bp
E0240+lva909bp1147bp
R0010+E0240+lva1117bp1355bp

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

DNA Length
PartPart LengthLength in pSB1A2
I13507861bp1099bp
I13507+lva894bp1132bp
R0010+I13507+lva1102bp1340bp

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

DNA Length
PartPart LengthLength in pSB1A2
E0022762bp1000bp
E0022+B0015899bp1137bp
B0034+E0022+B0015919bp1157bp
R0010+B0034+E0022+B00151127bp1365bp

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

DNA Length
PartPart LengthLength in Plasmid
E0032762bp1000bp
E0032+B0015899bp1137bp
B0034+E0032+B0015919bp1157bp
R0010+B0034+E0032+B00151127bp1365bp

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

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

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

3.PCR to make the whole parts GFPlva and RFPlva.

4.Run the gel. Team:NYMU-Taipei/GEL 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.

3154.jpg

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

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

3160.jpg

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

3161.jpg

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

templateforward primerreverse primer
positiveRFPVF2VR
YFP front partpLac+RBS+YFPlva+TermVF2XFP lva remover rp
YFP back partpLac+RBS+YFPlva+TermXFP lva remover fpVR
CFP front partpLac+RBS+CFPlva+TermVF2XFP lva remover rp
CFP back partpLac+RBS+CFPlva+TermXFP lva remover fpVR

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


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