Template:Team:UC Berkeley/Notebook/DV construction

From 2008.igem.org

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Construction of [<Phns>] Basic Part K112701
Construction of [<Phns>] Basic Part K112701
-
PCR dv001/dv006 on Phns from E. coli K12 MG1655            (150 bp, gp = A)
+
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 dv005/dv003 on Phns from E. coli K12 MG1655            (225 bp, gp = B)
-
PCR dv004/dv002 on Phns from E. coli K12 MG1655            (342 bp, gp = C)
+
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/dv003 on A+B              (355 bp, gp = D)
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dv001 Forward Biobricking of [<Phns>]      CgATAgaattcATGagatctGAAATATAGCTGTGCCATCAGCC
dv001 Forward Biobricking of [<Phns>]      CgATAgaattcATGagatctGAAATATAGCTGTGCCATCAGCC
dv002 Reverse Biobricking of [<Phns>]      cagtcggatccGCACGAAGAGTACGGATG
dv002 Reverse Biobricking of [<Phns>]      cagtcggatccGCACGAAGAGTACGGATG
-
dv003  Removing the 2nd EcoRI site in [<Phns>] (R)  GCCAGGAATGTAAGGAATTCAAAATTGTTC
+
dv003  Removing the 2nd EcoRI site in [<Phns>] (R)  GCCAGGAATGTAAGGgATTCAAAATTGTTC
dv004  Removing the 2ND EcoRI site in [<Phns>] (F)  GAACAATTTTGAATcCCTTACATTCCTGGC
dv004  Removing the 2ND EcoRI site in [<Phns>] (F)  GAACAATTTTGAATcCCTTACATTCCTGGC
dv005  Removing the FIRST EcoRI site in [<Phns>] (F)CGCTTAATAGGGgATTCTCGTAAACAC
dv005  Removing the FIRST EcoRI site in [<Phns>] (F)CGCTTAATAGGGgATTCTCGTAAACAC
dv006  Removing the FIRST EcoRI site in [<Phns>] (R)GTGTTTACGAGAATcCCCTATTAAGCG
dv006  Removing the FIRST EcoRI site in [<Phns>] (R)GTGTTTACGAGAATcCCCTATTAAGCG
-
</pre>
 
-
 
-
==K112702==
 
-
<pre>
 
-
Construction of <His-Tag! Basic Part K112702
 
-
 
-
Mixing of dv007/dv008  (27bp, EcoRI/BamHI/DpnI)
 
-
Sub into pBca1256                      (EcoRI/BamHI, 2472+27, L)
 
-
Product is pBca1256-K112702
 
-
-----------------------------------------------
 
-
dv007  Forward biobricking of <His-Tag!    CATCATCATCATCATCATtaaGGATCC
 
-
dv008  Reverse biobriquing of <His-Tag!    GGATCCttaATGATGATGATGATGATG
 
</pre>
</pre>
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Construction of S-tag> basic part K112705
Construction of S-tag> basic part K112705
-
Mix dv013/dv014 on pBca1              (47 bp, EcoRI/BamHI/DpnI)
+
Wobble PCR dv013/dv014               (47 bp, EcoRI/BamHI/DpnI)
Sub into pBca1256                      (EcoRI/BamHI, 2472+47, L)
Sub into pBca1256                      (EcoRI/BamHI, 2472+47, L)
Product is pBca1256-K112705
Product is pBca1256-K112705
Line 78: Line 66:
cgttaGGATCCgctgtccatgtgctggcgttcgaatttagcagcagcgg
cgttaGGATCCgctgtccatgtgctggcgttcgaatttagcagcagcgg
</pre>
</pre>
 +
==K112706==
==K112706==
<pre>
<pre>
Line 112: Line 101:
Construction of <PfhuA> basic part K112708
Construction of <PfhuA> basic part K112708
-
PCR of dv019/dv020 on  pBjh1205         (225bp, EcoRI/BamHI/DpnI)
+
PCR of dv019/dv020 on  MG1655         (225bp, EcoRI/BamHI/DpnI)
Sub into pBca1256                        (EcoRI/BamHI, 2472+225, L)
Sub into pBca1256                        (EcoRI/BamHI, 2472+225, L)
Product is pBca1256-K112708
Product is pBca1256-K112708
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Construction of <Bca1041> basic part K112710
Construction of <Bca1041> basic part K112710
-
PCR of dv023/dv024  Bca1041                  (122bp, EcoRI/BamHI/DpnI)
+
PCR of dv023/dv024  pSB1AG0-Bca1041                  (122bp, EcoRI/BamHI/DpnI)
Sub into pBca1256                      (EcoRI/BamHI, 2472+122, L)
Sub into pBca1256                      (EcoRI/BamHI, 2472+122, L)
Product is pBca1256-K112710
Product is pBca1256-K112710
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dv024 Reverse biobricking of Bca1041
dv024 Reverse biobricking of Bca1041
cgttaGGATCCatttcttttgggtatagcgtcgtgg
cgttaGGATCCatttcttttgggtatagcgtcgtgg
 +
 +
</pre>
 +
 +
==K112711==
 +
<pre>
 +
Construction of rbs.SpvR! basic part K112711
 +
 +
PCR of rbs.SpvR! al0027 and al0028  (933bp, EcoRI, BamHI, DpnI)
 +
Sub into pBca1256                        (EcoRI/BamHI, 2472+933bp, L)
 +
Product is pBca1256-K112711
 +
 +
------------------------------------------------
 +
al0027 Forward Biobricking of rbs.spvR! CCATAGAATTCatgAGATCTAAAAAAGGAGATATTATGGATTTCTTG
 +
 +
al0028 Reverse Biobricking of rbs.spvR! CGttaGGATCCaggtcagaaggtggactg
 +
 +
</pre>
 +
 +
==K112703 again==
 +
<pre>
 +
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
 +
</pre>
</pre>

Latest revision as of 21:57, 7 July 2008

Contents


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   (933bp, EcoRI, BamHI, DpnI)
Sub into pBca1256                        (EcoRI/BamHI, 2472+933bp, 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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