Template:Team:UC Berkeley/Notebook/DV construction

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K112701

Construction of [<Phns>] Basic Part K112701

PCR dv001/dv006 on Phns from E. coli K12 MG1655            (~342 bp, gp = A)
PCR dv005/dv003 on Phns from E. coli K12 MG1655            (225 bp, gp = B)
PCR dv004/dv002 on Phns from E. coli K12 MG1655            (~149 bp, gp = C)
---------------------------------------------------
PCR dv001/dv003 on A+B              (355 bp, gp = D)
---------------------------------------------------
PCR dv001/dv002 on C+D              (697bp, EcoRI/BamHI/DpnI)
Sub into pBca1256                   (EcoRI/BamHI, 2472+697)
Product is pBca1256-K112701
-----------------------------------------------
dv001	Forward Biobricking of [<Phns>]      CgATAgaattcATGagatctGAAATATAGCTGTGCCATCAGCC
dv002	Reverse Biobricking of [<Phns>]      cagtcggatccGCACGAAGAGTACGGATG
dv003  Removing the 2nd EcoRI site in [<Phns>] (R)  GCCAGGAATGTAAGGgATTCAAAATTGTTC
dv004  Removing the 2ND EcoRI site in [<Phns>] (F)  GAACAATTTTGAATcCCTTACATTCCTGGC
dv005  Removing the FIRST EcoRI site in [<Phns>] (F)CGCTTAATAGGGgATTCTCGTAAACAC
dv006  Removing the FIRST EcoRI site in [<Phns>] (R)GTGTTTACGAGAATcCCCTATTAAGCG

K112703

Construction of His-Tag> basic part K112703

Mix dv009/dv010                        (41bp, EcoRI/BamHI/DpnI)
Sub into pBca1256                      (EcoRI/BamHI, 2472+41, L)
Product is pBca1256-K112703
-----------------------------------------------
dv009	Forward Biobricking of His-Tag>
cgataGAATTCatgAGATCTatgCATCATCATCATCATCATGGATCCtaacg

dv010   Reverse Biobricking of His-Tag>
cgttaGGATCCATGATGATGATGATGATGcatAGATCTcatGAATTCtatgg

K112704

Construction of <S-tag! basic part K112704

Wobble PCR of dv011/dv012		(60 bp, EcoRI/BamHI/DpnI)
Sub into pBca1256			(EcoRI/BamHI)
Product is pBca1256-K112704
----------------------------------------
dv011		Forward Biobricking of <S-Tag!
cgataGAATTCatgAGATCTaaagaaaccgctgctgctaaattcgaacgcc

dv012		Reverse Biobricking of <S-Tag!
cgttaGGATCCttagctgtccatgtgctggcgttcgaatttagcagc

K112705

Construction of S-tag> basic part K112705

Wobble PCR dv013/dv014                (47 bp, EcoRI/BamHI/DpnI)
Sub into pBca1256                      (EcoRI/BamHI, 2472+47, L)
Product is pBca1256-K112705
-----------------------------------------------
dv013	Forward Biobricking of S-Tag>
cgataGAATTCatgAGATCTatgaaagaaaccgctgctgctaaattcg
dv014   Reverse Biobricking of S-Tag>
cgttaGGATCCgctgtccatgtgctggcgttcgaatttagcagcagcgg

K112706

Construction of <Pspv2> basic part K112706

PCR of dv015/dv016 on pBca1037          (500bp, EcoRI/BamHI/DpnI)
Sub into pBca1256                       (EcoRI/BamHI, 2472+ 500, L)
Product is pBca1256-K112706
-----------------------------------------------
dv015 Forward biobricking of <Pspv2>
cgataGAATTCatgAGATCTccttatgcagcgtaagggccgcaac

dv016 Reverse biobricking of <Pspv2>
cgttaGGATCCGATAATGTtTGCAGGGGAATTATTTTG

K112707

Construction of <Pspv> basic part K112707

PCR of dv017/dv016 on pBca1037         (499bp, EcoRI/BamHI/DpnI)
Sub into pBca1256                       (EcoRI/BamHI, 2472+499, L)
Product is pBca1256-K112707
-----------------------------------------------
dv017 Forward biobricking of <Pspv>
cgataGAATTCatgAGATCTaGATCCTGTGATGTTTGGCG

dv016 Reverse biobricking of <Pspv>
cgttaGGATCCGATAATGTtTGCAGGGGAATTATTTTG

K112708

Construction of <PfhuA> basic part K112708

PCR of dv019/dv020 on   MG1655          (225bp, EcoRI/BamHI/DpnI)
Sub into pBca1256                        (EcoRI/BamHI, 2472+225, L)
Product is pBca1256-K112708
-----------------------------------------------
dv019 Forward biobricking of <PfhuA>      
cgataGAATTCatgAGATCTgctaagcgtgaaataccggatgg

dv020 Reverse biobricking of <PfhuA>
cgttaGGATCCactctgatgtaaagtgaatgataacg

K112709

Construction of <b1006> basic part K112709
 
Wobble PCR of dv021/dv022              (66bp, EcoRI/BamHI/DpnI)
Sub into pBca1256                      (EcoRI/BamHI, 2472+66, L)
Product is pBca1256-K112709
-----------------------------------------------
dv021 Forward biobricking of b1006
cgataGAATTCatgAGATCTaaaaaaaaaccccgcccctgacagggcgg

dv022 Reverse biobricking of b1006
GGATCCtaaaaaaaaccccgccctgtcaggggcggggtttttt

K112710

Construction of <Bca1041> basic part K112710

PCR of dv023/dv024  pSB1AG0-Bca1041                   (122bp, EcoRI/BamHI/DpnI)
Sub into pBca1256                      (EcoRI/BamHI, 2472+122, L)
Product is pBca1256-K112710
-----------------------------------------------
dv023 Forward biobricking of Bca1041
cgataGAATTCatgAGATCTgCcggcttatcGgtcagtttc


dv024 Reverse biobricking of Bca1041
cgttaGGATCCatttcttttgggtatagcgtcgtgg

K112711

Construction of rbs.SpvR! basic part K112711

PCR of rbs.SpvR! al0027 and al0028   [template]
Sub into pBca1256                        (EcoRI/BamHI, 2472+(SpvR length), L)
Product is pBca1256-K112711

------------------------------------------------
al0027 Forward Biobricking of rbs.spvR! CCATAGAATTCatgAGATCTAAAAAAGGAGATATTATGGATTTCTTG

al0028 Reverse Biobricking of rbs.spvR! CGttaGGATCCaggtcagaaggtggactg

K112703 again

Construction of His-tag> basic part K112703, 2nd attempt

EIPCR of His-tag> dv025 and ca1246R on pBca1256   (2704bp EcoRI/BamHI,) 
Cyclize pBca1256-K112703               (EcoRI)
Product is pBca1256-K112703

dv025   Forward Biobricking of His-tag>  ctagtGAATTCatgGGATCCtaaCTCGAcgtgcagg
ca1246R Reverse EIPCR from mRFP to facilitate Biobricking of His-tag>  GACCTTCACCTTCACCTTCG
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