Construct worked with |
Things done |
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May 18th - May 22nd |
Preparations Plates |
Making Cm plates
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Stored in +4°C
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Preparations Competent Cells |
Preparing solutions for competent cells
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Stored in +4°C
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o/n culture of E. coli TOP10
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Chemical competent cells protocol
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Tranformation efficiency kit
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o/n culture of E. coli BL21(DE3)
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Chemical competent cells protocol
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Tranformation efficiency kit
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Colonies are the wrong color
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Preparations Propagations |
Transformed AH001-AH008 into TOP10
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AH001-AH004 & AH006
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Stored in +4°C
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No colonies for AH005, AH007 or AH008
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Construct worked with |
Things done |
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May 25th - May 29th |
Preparations Propagating |
Transformed AH007-AH009, AH005 & AH019 into TOP10
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AH007-AH009 & AH019
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o/n cultures
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MiniPrep
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o/n culture of AH001-AH004 & AH006
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MiniPrep
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No colonies for AH005
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Re-transform into TOP10
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Plates in rt over weekend
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Checking Competent Cells |
o/n culture of BL21(DE3) transformants
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MiniPrep
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PCR with VF2 & VR
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Smeared gels
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AH001+ AH003+AH008 -> AH013
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Repeat ligation
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Restrict & extract from gel
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Transformation into TOP10
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Plates in rt over weekend
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Testing RBS's Assembly |
Restrict AH001-AH004 & AH006-AH008
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Ligation
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AH002+ AH004+AH008 -> AH014
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Repeat ligation
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Restrict & extract from gel
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Transformation into TOP10
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Plates in rt over weekend
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AH001+ AH006+AH008 -> AH011
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Repeat ligation
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Restrict & extract from gel
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Transformation into TOP10
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Plates in rt over weekend
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Construct worked with |
Things done |
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June 22nd - June 26th |
Testing RBS's Assembly |
Re-assembling AH016 & AH018
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Transform into BL21(DE3)
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No colonies
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Testing RBS's Propagation |
o/n cultures of AH016 & AH018 in TOP10
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MiniPrep
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Transform into BL21(DE3)
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Plates in rt over the weekend
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Testing RBS's Measurement |
AH018 in BL21(DE3)
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o/n culture
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IPTG induction & blue chromoprotein measurement
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No blue chromoprotein
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Refresh on IPTG plate
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No blue chromoprotein
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Preparations Linearized Backbones |
o/n cultures of AH007-AH009
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MiniPrep
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Restrict & extract from gel
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Construct worked with |
Things done |
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July 13th - July 17th |
Preparations Propagations |
o/n culture of AH029
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MiniPrep
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CAR2 OK
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PCR purification
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Propane Plasmids 1&2 Construction |
PCR for CAR1-3 & AtoB1-2
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CAR1,3 & AtoB1-2 unspecific bands
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Gradient PCR for CAR3 & 4
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PCR with DMSO for CAR1, 3 & AtoB1, 2
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dunnowhat
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GFP Brick Construction |
o/n culture of AH044
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MiniPrep
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Propane Plasmid 2 Construction |
PCR for AtoB3-4
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Unspecific bands
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AtoB 4 extracted from gel
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Low concentration
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Gradient PCR for AtoB3
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Extract & purify from gel
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Construct worked with |
Things done |
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July 27th - July 31st |
Propane Plasmid 1 Construction |
Continue CAR1 colony screening
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Screen more colonies
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PCR purification
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PCR for new CAR2
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Gradient PCR with KAPA polymerase
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PCR purification
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Preparations Linearized Backbones |
Re-restrict AH043
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Gel purification
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Low yield
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Re-restrict
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Propagations |
o/n culture of AH031
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MiniPrep
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Transform AH033 into TOP10
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o/n culture
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MiniPrep
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Low yield
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o/n culture
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MiniPrep
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Low yield, but stored in -20°C
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Propane Plasmid 2 Construction |
TOPO Blunt End Cloning for AtoB1 and transformation into TOP10
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Screening with colony PCR
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PCR purification
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Cellulose Plasmid Construction |
PCR for Cen1 & Cen2
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Gradient PCR with KAPA polymerase
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PCR purification for Cen2
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Gel purification for Cen1
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2nd PCR for BglX (from AH021)
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Stored in -4°C over the weekend
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Propane Plasmid 1 Construction |
Re-do colony PCR for CAR3 successful TOPO colony
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Re-do colony PCR
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o/n culture of the colony
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MiniPrep
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PCR
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Violacein Plasmid Construction |
2nd PCR for VioA (from AH020)
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Stored in -4°C over the weekend
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KAPA gradient PCR for AtoB3,4 & CAR3
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Gel purification for AtoB4 & Car3
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PCR purification for AtoB3
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