SBB10Ntbk-JennaKelleher: Difference between revisions
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[[SBB10Ntbk-JennaKelleher3| 2/22/2010]]<br> | |||
[[SBB10Ntbk-JennaKelleher2| 2/17/2010]]<br> | [[SBB10Ntbk-JennaKelleher2| 2/17/2010]]<br> | ||
[[SBB10Ntbk- | ===Labwork=== | ||
Prepared sbb28 and sbb29 for PCR after creating a master mix with [[SBB10Ntbk-Jeni_Lee | Jeni Lee]]<br> | |||
sbb28 in 2k55 and sbb29 in 55 | |||
===Updated Constuction Files=== | |||
Biobricking of 099 rep protein basic part sbb29 | |||
PCR repo99-F and JG002-R on pEC52 gene (978 bp, EcoRI/BamHI) | |||
Sub into pBca9523-Bca1144 (EcoRI/BamHI, 910+2472, L) | |||
Product is pBca9523-sbb29 {rbs.repO99!} | |||
---------------------------- | |||
repo99-F Cloning of repo99 | |||
ccaaaGAATTCatgAGATCTcctggaaggaatcgtaacac | |||
JG002-R Cloning of O99 rep Gene | |||
CTGATGGATCCCTACTCTACAAGACCTCGTTTTttc | |||
Biobricking of CA42 rep gene basic part sbb28 | |||
PCR repCA42-F and KRM002 on pEC22-CA42 gene (1496 bp, EcoRI/BamHI) | |||
Sub into pBca9523-Bca1144 (EcoRI/BamHI, 910+2472, L) | |||
Product is pBca9523-sbb28 {5'UTR.repCA42!} | |||
---------------------------- | |||
repCA42-F Cloning of repCA42 | |||
ccaaaGAATTCatgAGATCTcagctgaagtgaccggattag | |||
KRM002 Reverse oligo for cloning of rbs.repCA42! | |||
ctgatGGATCCctattttccgcttttccag | |||
Biobricking of pre-pro sequence basic part sbb42 | |||
Eco/Bam transfer pBjh1601AC-Bjh1723 | |||
Sub into pBjk2741-Bca1144 (EcoRI/BamHI, 910+2170, L) | |||
Product is pBjk2741-Bjh1723 {rbs_pelB>} | |||
[[SBB10Ntbk-JennaKelleher1| 2/10/2010]]<br> | [[SBB10Ntbk-JennaKelleher1| 2/10/2010]]<br> | ||
===Project Information=== | ===Project Information=== |
Revision as of 12:44, 24 February 2010
Labwork
Prepared sbb28 and sbb29 for PCR after creating a master mix with Jeni Lee
sbb28 in 2k55 and sbb29 in 55
Updated Constuction Files
Biobricking of 099 rep protein basic part sbb29
PCR repo99-F and JG002-R on pEC52 gene (978 bp, EcoRI/BamHI) Sub into pBca9523-Bca1144 (EcoRI/BamHI, 910+2472, L) Product is pBca9523-sbb29 {rbs.repO99!} ---------------------------- repo99-F Cloning of repo99 ccaaaGAATTCatgAGATCTcctggaaggaatcgtaacac JG002-R Cloning of O99 rep Gene CTGATGGATCCCTACTCTACAAGACCTCGTTTTttc
Biobricking of CA42 rep gene basic part sbb28
PCR repCA42-F and KRM002 on pEC22-CA42 gene (1496 bp, EcoRI/BamHI) Sub into pBca9523-Bca1144 (EcoRI/BamHI, 910+2472, L) Product is pBca9523-sbb28 {5'UTR.repCA42!} ---------------------------- repCA42-F Cloning of repCA42 ccaaaGAATTCatgAGATCTcagctgaagtgaccggattag KRM002 Reverse oligo for cloning of rbs.repCA42! ctgatGGATCCctattttccgcttttccag
Biobricking of pre-pro sequence basic part sbb42
Eco/Bam transfer pBjh1601AC-Bjh1723 Sub into pBjk2741-Bca1144 (EcoRI/BamHI, 910+2170, L) Product is pBjk2741-Bjh1723 {rbs_pelB>}
Project Information
29071-O99 rep Protein, CA42 rep Gene, Pre-Pro sequence
Construction Files
Jenna Kelleher 099 Rep Protein (sbb29)
Jenna Kelleher CA42 Rep Gene (sbb28)
Jenna Kelleher Pre-pro Sequence (sbb42)
Useful Protocols
For SBB28 and SBB29:
Cloning by PCR
Regular Zymo Cleanup
For SBB42:
Eco-Bam Transfer
Small-Frag Zymo Cleanup
For All 3:
EcoRI/BamHI Digest of PCR Products
Zymo Gel Purification
Ligation of EcoRI/BamHI digests
Transformation by heat-shock
Picking of colonies
Macherey-Nagel Miniprep
EcoRI/BamHI Digest of PCR Products
Analytical digests (Mapping)