Team:Lethbridge/Notebook

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(Difference between revisions)
(August)
(September)
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== September ==
 
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===September 3===
 
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'''Roxanne, Ashley, Kirsten'''
 
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Made 1.5L agar LB media according to lab protocol.  Roxanne is autoclaving tomorrow.
 
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Pick 3 lumazine-dT colonies from glycerol stocks to grow overnight
 
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Restriction Digest
 
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Reaction 1
 
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{|
 
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|
 
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|'''Ribo-cheZ-dT'''
 
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|''GFP-dT''
 
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|sRBS-mmS6
 
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|'''Mr. Gene mmS6'''
 
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|-
 
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|milliQ water
 
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|'''5 µL'''
 
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|''5 µL''
 
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|5 µL
 
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|'''5 µL'''
 
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|-
 
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|Buffer tango
 
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|'''3 µL'''
 
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|''3 µL''
 
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|3 µL
 
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|'''3 µL'''
 
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|-
 
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|DNA
 
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|'''20 µL'''
 
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|''20 µL''
 
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|20 µL
 
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|'''20 µL'''
 
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|-
 
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|XbaI
 
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|'''1 µL'''
 
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|''1 µL''
 
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|1 µL
 
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|'''1 µL'''
 
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|-
 
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|PstI
 
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|'''1 µL'''
 
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|''1 µL''
 
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|1 µL
 
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|'''1 µL'''
 
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|-
 
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|total
 
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|'''30 µL'''
 
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|''30 µL''
 
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|30 µL
 
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|'''30 µL'''
 
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|}
 
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Reaction 2
 
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{|
 
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|
 
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|'''Ribo-cheZ-dT'''
 
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|''GFP-dT''
 
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|sRBS-mmS6
 
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|'''Mr. Gene mmS6'''
 
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|-
 
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|milliQ water
 
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|'''3 µL'''
 
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|''3 µL''
 
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|3 µL
 
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|'''3 µL'''
 
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|-
 
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|Buffer tango
 
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|'''1 µL'''
 
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|''1 µL''
 
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|1 µL
 
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|'''1 µL'''
 
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|-
 
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|DNA
 
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|'''5 µL'''
 
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|''5 µL''
 
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|5 µL
 
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|'''5 µL'''
 
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|-
 
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|EcoRI
 
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|'''1 µL'''
 
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|''1 µL''
 
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|1 µL
 
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|'''1 µL'''
 
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|-
 
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|total
 
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|'''10 µL'''
 
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|''10 µL''
 
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|10 µL
 
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|'''10 µL'''
 
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|}
 
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Into HWB at 37°C for 2 hours
 
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Gel Electrophoresis (concentration):
 
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Lane
 
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# 1kb ladder
 
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# ribo-cheZ-dT (EcoRI)
 
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#GFP-dT (EcoRI)
 
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#sRBS-mmS6-dT (EcoRI)
 
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#NEYFP (XbaI,PstI)
 
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#NEYFP (ecoRI, speI)
 
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#NCFP (ecoRI, speI)
 
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#NECFP (xbaI,pstI)
 
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#CCFP(ecoRI,speI)
 
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#CECFP(ecoRI,speI)
 
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#CCFP (notI,pstI)
 
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#NEYFP (ecoRI,speI)
 
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#CECFP (xbaI)
 
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#CEYFP(xbaI,pstI)
 
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#NCFP (xbaI,pstI)
 
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#CEYFP(xbaI)
 
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#pLacI
 
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#pStrong
 
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#1kb ladder
 
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5µL sample, 1 µL loading dye (6x)
 
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6 µL ladder
 
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[[Image:September_3_concentration_gel.png|250px]]
 
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Ran at 100V for 1 hour
 
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Concentrations good!
 
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'''Alix'''
 
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Gel extraction of:
 
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sRBS-mmS6-dT
 
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riboswitch-cheZ-dT
 
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GFP-dT
 
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mmS6
 
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Following Qiagen benchtop protocol
 
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===September 4===
 
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'''Ashley'''
 
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Ligation of:
 
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*C-term-dT +C-CFP #2
 
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*C-term-dT +C-ECFP #2
 
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*C-term-dT + C-EYFP #2
 
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*pSB1A3-2+C-CFP#1
 
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*pSB1A3-2+C-ECFP#1
 
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*pSB1A3-2+C-EYFP#1
 
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*mRBS-N-term +N-CFP#1
 
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*mRBS-N-term +N-EYFP#1
 
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*mRBS-N-term +N-ECFP#1
 
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*pSB1A3-1+N-CFP#2
 
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*pSB1A3-1+N-ECFP#2
 
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*pSB1A3-1+N-EYFP#2
 
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Reaction
 
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{|
 
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|
 
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|'''Control'''
 
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|All reactions
 
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|-
 
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|milliQ water
 
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|'''7.5 µL'''
 
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|4.5 µL
 
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|-
 
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|10x buffer T4
 
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|'''1 µL'''
 
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|1 µL
 
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|-
 
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|Vector
 
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|'''1 µL'''
 
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|1 µL
 
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|-
 
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|Insert
 
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|'''0 µL'''
 
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|3 µL
 
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|-
 
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|T4 DNA ligase
 
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|'''0.5 µL'''
 
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|0.5 µL
 
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|-
 
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|total
 
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|'''10 µL'''
 
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|10 µL
 
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|}
 
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Restriction Digest
 
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{|
 
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|
 
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|Control (no enzyme)
 
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|'''Lumazine-dT (1)'''
 
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|-
 
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|milliQ water
 
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|2.4 µL
 
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|'''0.4 µL'''
 
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|-
 
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|10x buffer Tango
 
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|1.6 µL
 
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|'''1.6 µL'''
 
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|-
 
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|DNA
 
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|16 µL
 
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|'''16 µL'''
 
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|-
 
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|XbaI
 
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|0 µL
 
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|'''1 µL'''
 
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|-
 
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|PstI
 
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|0 µL
 
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|'''1 µL'''
 
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|-
 
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|total
 
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|20 µL
 
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|'''20 µL'''
 
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|}
 
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</head>
 
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<body>
 
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<ul id="qm0" class="qmmc">
 
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<li><a href="https://2009.igem.org/Team:Lethbridge">Home</a></li>
 
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<li><a href="https://2009.igem.org/Team:Lethbridge/Notebook">Top of Page</a></li>
 
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<li class="qmclear">&nbsp;</li></ul>
 
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<script type="text/javascript">qm_create(0,false,0,500,false,false,false,false,false);</script>
 
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</body>
 
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===September 8===
 
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'''Ashley'''
 
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Made 1.5L LB agar according to protocol
 
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Made 300mL LB broth according to protocol
 
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Gel Electrophoresis (for extraction)
 
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5 µL ladder
 
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5 µL sample, 1 µL dye
 
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Lane
 
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# 1 kb ladder
 
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#empty^
 
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#empty
 
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# lumazine-dT (XbaI, PstI) 600bp
 
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^there was a control (no enzyme) but not enough DNA to load
 
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Ran at 1000V for one hour
 
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Gel extraction according to Qiagen benchtop protocol
 
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===Sept 10===
 
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'''Ashley, Kirsten'''
 
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Ran 1% agarose
 
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Lane
 
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# 1 kb ladder
 
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#C-term-dT + C-EYFP #2
 
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#pSB1A3-1+N-CFP#2
 
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#pSB1A3-1+N-ECFP#2
 
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#lumazine+sRBS
 
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#pSB1A3-2+C-EYFP#1
 
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#pSB1A3-1+N-EYFP#2
 
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#mRBS-N-term +N-CFP#1
 
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#mmS6+pSB1A3-1
 
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#GFP-dT
 
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#mmS6-dT
 
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#pSB1A3-2+C-ECFP#1
 
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#mRBS-N-term +N-ECFP#1
 
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#mRBS-N-term +N-EYFP#1
 
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#C-term-dT +C-CFP #2
 
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#C-term-dT +C-ECFP #2
 
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#pSB1A3-2+C-CFP#1
 
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3 µL sample + 0.5 µL dye
 
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6 µL 1 kb ladder
 
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'''Mackenzie'''
 
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Transformation into DH5α
 
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*sRBS-lumazine-dT (km)
 
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*mmS6-dT (km)
 
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*pSB1A3+mmS6
 
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*mRBS-N-term +N-ECFP#1
 
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*mRBS-N-term +N-EYFP#1
 
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*C-term-dT +C-ECFP #2
 
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*C-term-dT + C-EYFP #2
 
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*pSB1A3-2+C-ECFP#1
 
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*pSB1A3-2+C-EYFP#1
 
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The following were not transformed due to lack of competent cells:
 
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*pSB1A3-1+N-ECFP#2
 
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*pSB1A3-1+N-EYFP#2
 
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*GFP-dT
 
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<html>
 
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<head>
 
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<style type="text/css">
 
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/* QuickMenu 0 */
 
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ul#qm0 ul
 
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</head>
 
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<body>
 
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<ul id="qm0" class="qmmc">
 
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<li><a href="https://2009.igem.org/Team:Lethbridge">Home</a></li>
 
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<li><a href="https://2009.igem.org/Team:Lethbridge/Notebook">Top of Page</a></li>
 
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<li class="qmclear">&nbsp;</li></ul>
 
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<script type="text/javascript">qm_create(0,false,0,500,false,false,false,false,false);</script>
 
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</body>
 
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</html>
 
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===September 14===
 
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'''Ashley'''
 
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PCR of EYFP, CFP and ECFP
 
-
 
-
See Aug 6 for volumes
 
-
 
-
CFP(1)=prefix primer and antisense C-term suffix
 
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CFP(2)=N-term prefix and antisense suffix
 
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EYFP(1)=prefix primer and antisense C-term suffix
 
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EYFP(2)=N-term prefix and antisense suffix
 
-
 
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ECFP(1)=prefix primer and antisense C-term suffix
 
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ECFP(2)=N-term prefix and antisense suffix
 
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-
Cycle: tail67
 
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-
===Sept 16===
 
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-
'''Ashley'''
 
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-
Miniprep of mmS6-dT 1,2 and 3.  Used Qiagen benchtop protocol and kit.  Eluted with 50 µL buffer EB. Put into -20°C freezer orange tray.
 
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PCR Purification of restriction digest of PCR products.
 
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-
 
-
===September 24===
 
-
'''Ashley, Kirsten'''
 
-
 
-
1% gel electrophoresis
 
-
6uL ladder (1kb)
 
-
5uL sample +1 uL 6x loading dye
 
-
100V, 40min
 
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Lane
 
-
#1kb Ladder
 
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#C-CFP-1
 
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#C-ECFP-1
 
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#N-EYFP-2
 
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#N-CFP-2
 
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#N-ECFP-2
 
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#C-EYFP-1
 
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#CFP-1 Purified PCR
 
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#ECFP-1 Purified PCR
 
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#EYFP-2 Purified PCR
 
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#CFP-2 Purified PCR
 
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#ECFP-1 Purified PCR
 
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#EYFP-1 Purified PCR
 
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#C-CFP(1)
 
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#ECFP(1)
 
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#C-EYFP(1)
 
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#N-CFP(2)
 
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#N-EYCFP(2)
 
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#N-EYFP(2)
 
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#1KB LADDER
 
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[[Image:Sept_24_-_control_for_PCR.png|250px]]
 
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Gel was run to check for DNAses, found that DPN-1 had DNAses.
 
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===Sept 25===
 
-
1% agarose gel 100V, 40min
 
-
 
-
Lane:
 
-
#1kb ladder
 
-
#mms6(EcoRI,SpeI)
 
-
#pLacI (SpeI, PstI)
 
-
#Lumazine-dT
 
-
 
-
Only pLacI success, gell extracted and eluted with 50uL buffer EB.
 
-
 
-
Miniprep of:
 
-
*3x mmS6-dT
 
-
*3x pSB1A3-mmS6
 
-
*2x cheZ-dT
 
-
*1x riboswitch-cheZ-dT
 
-
*2x GFP-dT
 
-
*3x C-EYFP-dT
 
-
 
-
Restricted all of the above with EcoRI
 
-
*8uL DNA
 
-
*1uL 10x buffer tango
 
-
*1uL EcoRI
 
-
 
-
for 1 hour.
 
-
 
-
Restriction of fusion proteins with either EcoRI,SpeI or XbaI,PstI
 
-
*Heat inactivated a 60C for 10min
 
-
 
-
Ran on a gel (1% agarose)100V,40min
 
-
 
-
Lane:
 
-
#1kb ladder
 
-
#pSB-mmS6 #1
 
-
#pSB-mmS6 #2
 
-
#pSB-mmS6 #3
 
-
#mms6-dT #1
 
-
#mms6-dT #2
 
-
#mms6-dT #3
 
-
#EYFP C-term-dT #1
 
-
#EYFP C-term-dT #2
 
-
#EYFP C-term-dT #3
 
-
#GFP-dT#1
 
-
#GFP-dT#2
 
-
#Riboswitch-cheZ-dT
 
-
#cheZ-dT#1
 
-
#cheZ-dT#2
 
-
#C-CFP (EcoRI,SpeI)
 
-
#C-CFP (XbaI,PstI)
 
-
#C-ECFP (EcoRI,SpeI)
 
-
#C-ECFP (XbaI,PstI)
 
-
#1kb laddder
 
-
 
-
[[Image:Sept_25-first_lanes.png|250px|left]]
 
-
 
-
[[Image:Sept_25_far_lanes.png|250px|left]]
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
*Restriction of Fusion proteins with Dpn1
 
-
*Heat inactivated a 60C for 10min
 
-
 
-
Lane:
 
-
#1kB ladder
 
-
#N-ECFP
 
-
#N-ECFP
 
-
#N-EYFP
 
-
#E-EYFP
 
-
#N-CFP
 
-
#N-CFP
 
-
#C-EYFP
 
-
#C-EYFP
 
-
#1kB ladder
 
-
 
-
 
-
Restriction of Lumazine-dT
 
-
*30uL DNA
 
-
*4uL 10x buffer
 
-
*3uL EcoRI
 
-
*3uL SpeI
 
-
Setup Liquid culture of the Mr. Gene mmS6 x3
 
-
 
-
===September 28===
 
-
'''Ashley'''
 
-
 
-
1% agarose gel
 
-
*5uL DNA+1uL 6x loading dye
 
-
*6uL ladder (1kb)
 
-
 
-
Lane:
 
-
#1kb ladder
 
-
#N-ECFP (XbaI,PstI)
 
-
#N-ECFP (EcoRI, SpeI)
 
-
#N-EYFP(XbaI,PstI)
 
-
#N-EYFP(EcoRI, SpeI)
 
-
#N-CFP(XbaI,PstI)
 
-
#N-CFP(EcoRI, SpeI)
 
-
#C-EYFP(XbaI,PstI)
 
-
#C-EYFP(EcoRI, SpeI)
 
-
#1kb ladder
 
-
 
-
Ran at 100V for 40 min
 
-
 
-
[[Image:September_28th.jpg|250px]]
 
-
<html>
 
-
<head>
 
-
 
-
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== October ==
== October ==

Revision as of 19:02, 21 October 2009


Igembanner.jpg







October

October 1

Roxanne Gel for gel extraction

Restriction Ribo-Chez-dT

  • 2x CheZ-dT
  • 2x GFP-dT
  • 3x C-EYFP-dT

Ligation

  • C-ECFP-dT
  • C-CFP-dT
  • C-EYFP-dT
  • mRBS-N-ECFP
  • mRBS-N-EYFP
  • pSB-C-EFP
  • pSB-C-EYFP
  • pSB-C-CFP
  • pSB-N-ECFP
  • pSB-N-EYFP

Aliquoted DH5α cells

Ashley

Tube Tube + Gel Gel
1.1226g 1.4931g 0.2671g
1.012g 1.2302g 0.1290g

Eluted with 50uL buffer EB

Ligation

1:3 2:6
lum-dT 3uL 6uL
SRBS 1uL 2uL
Ligase Buffer 1uL 2uL
Water 4.5uL 9uL
Ligase 0.5uL 1uL

Oct 2

Pick Colonies:

  • sRBS + Lumazine-dT
  • mRBS + N-term FPs
  • C-term FPs + dT
  • pSB + N-term FPs
  • pSB + N-term FPs

Gel Extraction:

  • Riboswitch-cheZ-dT
  • 2x cheZ-dT (in case Ribo-cheZ-dT isn't actually there)
  • 3x C-EYFP-dT
  • 2x GFP-dT

Liquid Culture:

  • Riboswitch-cheZ-dT
  • 2x cheZ-dT (in case Ribo-cheZ-dT isn't actually there)
  • 3x C-EYFP-dT
  • 2x GFP-dT

Miniprep:

  • Mr. Gene mms6

Sat Oct 3

Miniprep:

  • sRBS + Lumazine-dT
  • mRBS + N-term FPs
  • C-term FPs + dT
  • pSB + N-term FPs
  • pSB + N-term FPs
  • sRBS + Lumazine-dT

Restriction:

Analytic and Prep:

  • mRBS + N-term FPs
  • C-term FPs + dT
  • pSB + N-term FPs
  • pSB + N-term FPs

Analytic only:

  • Mr. Gene mms6

Prep only:

  • pBAD-TetR

Oct 5

Gel extraction:

  • sRBS + Lumazine-dT
  • mRBS + N-term FPs
  • C-term FPs + dT
  • pSB + N-term FPs
  • pSB + N-term FPs

Ligation:

  • pStrong + Riboswitch-cheZ-dT
  • Riboswitch-GFP-dT
  • pLac + sRBS-Lumazine-dT
  • pBad-TetR + C-term FPs
  • pBad-TetR + N-term FPs
  • pTetR + N-term

Transformation:

  • pLac + sRBS-Lumazine-dT
  • pBad-TetR + C-term FPs
  • pBad-TetR + N-term FPs
  • pTetR + N-term ECFP
  • pStrong + Riboswitch-cheZ-dT
  • Riboswitch-GFP-dT

Sequencing:

  • sRBS + Lumazine-dT
  • mRBS + N-term FPs
  • C-term FPs + dT
  • pSB + N-term FPs
  • pSB + N-term FPs
  • Mr. Gene mms6

Tues Oct 6

Pick Colonies:

  • pLac + sRBS-Lumazine-dT
  • pBad-TetR + C-term FPs
  • pBad-TetR + N-term FPs
  • pTetR + N-term ECFP
  • pStrong + Riboswitch-cheZ-dT
  • Riboswitch-GFP-dT

Wed Oct 7

Miniprep:

  • pLac + sRBS-Lumazine-dT
  • pBad-TetR + C-term FPs
  • pBad-TetR + N-term FPs
  • pTetR + C-term ECFP
  • pTetR + N-term ECFP
  • pStrong + Riboswitch-cheZ-dT
  • Riboswitch-GFP-dT

Analytic Restriction:

  • pLac + sRBS-Lumazine-dT
  • pBad-TetR + C-term FPs
  • pBad-TetR + N-term FPs
  • pTetR + N-term ECFP
  • pStrong + Riboswitch-cheZ-dT
  • Riboswitch-GFP-dT

Prep Restriction:

  • Riboswitch-GFP-dT

Thurs Oct 8

Gel Extraction:

  • Riboswitch-GFP-dT

Ligation:

  • pStrong + Riboswitch-GFP-dT

Transformation:

  • pStrong + Riboswitch-GFP-dT

Sequencing:

  • pLac-sRBS-Lumazine-dT
  • pBad-TetR-C-term FPs
  • pBad-TetR-N-term FPs
  • pTetR-N-term ECFP
  • pStrong-Riboswitch-cheZ-dT
  • Riboswitch-GFP-dT

Maxiprep:

  • pLac-sRBS-Lumazine-dT
  • pBad-TetR-C-term FPs
  • pBad-TetR-N-term FPs
  • pTetR-N-term ECFP
  • pStrong-Riboswitch-GFP-dT

Restriction:

  • pLac-sRBS-Lumazine-

Analytic and Prep:

  • pBad-TetR-C-term FPs
  • pBad-TetR-N-term FPs
  • pTetR-N-term ECFP
  • C-term CFP (has pTetR)

Analytic Only:

  • pStrong-Riboswitch-GFP-dT

Fri Oct 9 Overexpression pLac + sRBS-Lumazine-dT Gel Extraction pLac-sRBS-Lumazine-dT pBad-TetR-C-term FPs pBad-TetR-N-term FPs pTetR-N-term ECFP C-term CFP (has pTetR) Motility Media

Oct 12

Ligation:

  • pLac-sRBS-Lumazine-dT + pBad-TetR-C-term EYFP
  • pLac-sRBS-Lumazine-dT + pBad-TetR-N-term EYFP

Transformation:

  • pLac-sRBS-Lumazine-dT + pBad-TetR-C-term EYFP
  • pLac-sRBS-Lumazine-dT + pBad-TetR-N-term EYFP

Plate Testing:

  • pStrong-Riboswitch-GFP-dT

Motility Test:

  • pStrong + Riboswitch-cheZ-dT

Oct 13

Pick Colonies

  • pLac-sRBS-Lumazine-dT + pBad-TetR-C-term EYFP
  • pLac-sRBS-Lumazine-dT + pBad-TetR-N-term EYFP

Analysis of Plate and Motility Tests

Wed Oct 14

Miniprep:

  • pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP
  • pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP

Restriction:

  • pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP
  • pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP

Thurs Oct 15

Gel Extraction:

  • pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP
  • pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP

Ligation:

  • pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP + C-term CFP
  • pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP + pTetR-N-term CFP

Transformation:

  • pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP + C-term CFP
  • pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP + pTetR-N-term CFP

Expression Testing:

  • pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP
  • pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP

Fri Oct 16

Pick Colonies:

  • pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP + C-term CFP
  • pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP + pTetR-N-term CFP

Sat Oct 17

Maxiprep:

  • pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP + C-term CFP
  • pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP + pTetR-N-term CFP