IGEM:Cambridge/2008/Notebook/Voltage/BioBrick Manipulation

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OsmY was PCRed out of the biobricks which contained it - BBa_J45996 (A) , BBa_J45995 (B) and BBa_J45250 (C). The primers were designed to have the BioBrick prefix and suffix, but in addition to have RBSs of 3 different strengths (B0030, B0031 and B0032) included in the suffix.
OsmY was PCRed out of the biobricks which contained it - BBa_J45996 (A) , BBa_J45995 (B) and BBa_J45250 (C). The primers were designed to have the BioBrick prefix and suffix, but in addition to have RBSs of 3 different strengths (B0030, B0031 and B0032) included in the suffix.
They were then cut with EcoRI and PstI and ligated into pSB4C5, which had been cut with the same enzymes. This was done at a 16:1 ratio of masses as OsmY is about 250bp and pSB4C5 is 4kbp. TOP10 cells were then transformed with the ligation product. The cells were then grown on chloramphenicol plates at 37°C and single colonies were then PCRed using standard BioBrick protocol.
They were then cut with EcoRI and PstI and ligated into pSB4C5, which had been cut with the same enzymes. This was done at a 16:1 ratio of masses as OsmY is about 250bp and pSB4C5 is 4kbp. TOP10 cells were then transformed with the ligation product. The cells were then grown on chloramphenicol plates at 37°C and single colonies were then PCRed using standard BioBrick protocol.
 +
==Primers==
==Primers==
-
OsmY Forward:        CTA TGA ATT CAT ATT CTA GAG CTG GCA CAG GAA CGT TAT CC                                      
+
OsmY Forward:        CTA TGA ATT CAT ATT CTA GAG CTG GCA CAG GAA CGT TAT CC    
-
OsmY-B0030 Reverse:  CGC GCT GCA GCT CTA CTA GTT TTC TCC TCT TTA ATT TGT TAA ATA TAG A                          
+
                                 
-
OsmY-B0031 Reverse:  CTC TCT GCA GCT CTA CTA GTG GTT TCC TGT GTG ATT GTT AAA TAT AGA T            
+
OsmY-B0030 Reverse:  CGC GCT GCA GCT CTA CTA GTT TTC TCC TCT TTA ATT TGT TAA ATA TAG A  
 +
                       
 +
OsmY-B0031 Reverse:  CTC TCT GCA GCT CTA CTA GTG GTT TCC TGT GTG ATT GTT AAA TAT AGA T  
 +
         
OsmY-B0032 Reverse:  CTC TCT GCA GCT CTA CTA GTC TTT CCT GTG TGA TTG TTA AAT ATA GAT CA             
OsmY-B0032 Reverse:  CTC TCT GCA GCT CTA CTA GTC TTT CCT GTG TGA TTG TTA AAT ATA GAT CA             
 +
=Kdp=
=Kdp=
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==Primers==
==Primers==
-
KDP Forward:  ATA TGA ATT CAT ATT CTA GAT GAG TGC AGG CGT GAT AAC CGG CGT ATT              
+
KDP Forward:  ATA TGA ATT CAT ATT CTA GAT GAG TGC AGG CGT GAT AAC CGG CGT ATT  
 +
           
KDP Reverse:  CTC TCT GCA GCT CTA CTA GTT TAT TCA TCA AGT TTA TCC AGC GCC AGA T             
KDP Reverse:  CTC TCT GCA GCT CTA CTA GTT TAT TCA TCA AGT TTA TCC AGC GCC AGA T             
 +
=InFusion=
=InFusion=
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==Primers==
==Primers==
-
Forward:  ATA TGA ATT CAT ATT CTA GAT GAG TGC AGG CGT GAT AAC CGG CGT ATT              
+
Forward:  ATA TGA ATT CAT ATT CTA GAT GAG TGC AGG CGT GAT AAC CGG CGT ATT  
 +
           
Reverse:  CTC TCT GCA GCT CTA CTA GTT TAT TCA TCA AGT TTA TCC AGC GCC AGA T
Reverse:  CTC TCT GCA GCT CTA CTA GTT TAT TCA TCA AGT TTA TCC AGC GCC AGA T

Revision as of 06:40, 28 October 2008

Contents

OsmY RBS

OsmY was PCRed out of the biobricks which contained it - BBa_J45996 (A) , BBa_J45995 (B) and BBa_J45250 (C). The primers were designed to have the BioBrick prefix and suffix, but in addition to have RBSs of 3 different strengths (B0030, B0031 and B0032) included in the suffix. They were then cut with EcoRI and PstI and ligated into pSB4C5, which had been cut with the same enzymes. This was done at a 16:1 ratio of masses as OsmY is about 250bp and pSB4C5 is 4kbp. TOP10 cells were then transformed with the ligation product. The cells were then grown on chloramphenicol plates at 37°C and single colonies were then PCRed using standard BioBrick protocol.


Primers

OsmY Forward: CTA TGA ATT CAT ATT CTA GAG CTG GCA CAG GAA CGT TAT CC

OsmY-B0030 Reverse: CGC GCT GCA GCT CTA CTA GTT TTC TCC TCT TTA ATT TGT TAA ATA TAG A

OsmY-B0031 Reverse: CTC TCT GCA GCT CTA CTA GTG GTT TCC TGT GTG ATT GTT AAA TAT AGA T

OsmY-B0032 Reverse: CTC TCT GCA GCT CTA CTA GTC TTT CCT GTG TGA TTG TTA AAT ATA GAT CA


Kdp

Kdp was PCRed out of the chromosome of MG1655. The primers were designed to have the BioBrick prefix and suffix. It was then cut with EcoRI and PstI and ligated into pSB4C5, which had been cut with the same enzyme. As Kdp is 4.4kb and the vector is 4kb approximatley the 1.1:1 ratio of mass was used (Kdp:pSB4C5). TOP10 cells were then transformed with the ligation product. The cells were then plated onto 35μg/mL chloramphenicol and grown at 37°C.

Primers

KDP Forward: ATA TGA ATT CAT ATT CTA GAT GAG TGC AGG CGT GAT AAC CGG CGT ATT

KDP Reverse: CTC TCT GCA GCT CTA CTA GTT TAT TCA TCA AGT TTA TCC AGC GCC AGA T


InFusion

InFusion primers were designed for both OsmY-RBS and KDP as well as the terminator containing plasmid B1002. These primers were designed to give the backbone with the terminator still attached.

Primers

Forward: ATA TGA ATT CAT ATT CTA GAT GAG TGC AGG CGT GAT AAC CGG CGT ATT

Reverse: CTC TCT GCA GCT CTA CTA GTT TAT TCA TCA AGT TTA TCC AGC GCC AGA T

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