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New Part Design(PCR)
From 2010.igem.org
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(New page: The following protocol is for creating a new biobricks part from a template DNA. '''Primer Design''' Design a forward primer to your new BioBrick part comprised of the BioBrick protein cod...) |
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| - | The following protocol is for creating a new biobricks part from a | + | The following protocol is for creating a new biobricks part from a protein coding sequence. |
'''Primer Design''' | '''Primer Design''' | ||
| - | Design a forward primer to your new BioBrick part comprised of the BioBrick protein coding prefix sequence GTT TCT TCG AAT TCG CGG CCG CTT CTA G followed by the first 20-30 nucleotides of the protein coding region, beginning with the ATG | + | |
| - | Design a reverse primer to your new BioBrick part comprised of the last 20-30 or so nucleotides of the protein coding sequence, excluding the stop codon, followed by the BioBrick protein coding suffix sequence 5'-TAA TAA TAC TAG TAG CGG CCG CTG CAG GAA GAA AC-3'. Note that the BioBrick protein coding suffix sequence includes the double stop codon TAA TAA. (Note: In some cases, for example if you wish use your part directly for making protein fusions using BioScaffold parts [1] or make your part more versatile by giving other users the capacity to do so in the future, then you should only include one stop codon TAA or make a version of your part that has only one stop codon. In this case your sequence should be followed by the BioScaffold and BioBrick compatible protein suffix 5'-TAA TAC TAG TAG CGG CCG CTG CAG GAA GAA AC-3' instead of the BioBrick protein coding suffix sequnce. Since the scar region that is created by the composition of parts, also introduces a stop codon after protein parts, including only one stop codon within the frame of the part may be sufficient (especially if your part will be followed by a subsequent part.)) | + | # Design a forward primer to your new BioBrick part comprised of the BioBrick protein coding prefix sequence GTT TCT TCG AAT TCG CGG CCG CTT CTA G followed by the first 20-30 nucleotides of the protein coding region, beginning with the ATG |
| - | Be sure and follow the guidelines above for design of the primer sequence that is complementary to the template. | + | # Design a reverse primer to your new BioBrick part comprised of the last 20-30 or so nucleotides of the protein coding sequence, excluding the stop codon, followed by the BioBrick protein coding suffix sequence 5'-TAA TAA TAC TAG TAG CGG CCG CTG CAG GAA GAA AC-3'. Note that the BioBrick protein coding suffix sequence includes the double stop codon TAA TAA. (Note: In some cases, for example if you wish use your part directly for making protein fusions using BioScaffold parts [1] or make your part more versatile by giving other users the capacity to do so in the future, then you should only include one stop codon TAA or make a version of your part that has only one stop codon. In this case your sequence should be followed by the BioScaffold and BioBrick compatible protein suffix 5'-TAA TAC TAG TAG CGG CCG CTG CAG GAA GAA AC-3' instead of the BioBrick protein coding suffix sequnce. Since the scar region that is created by the composition of parts, also introduces a stop codon after protein parts, including only one stop codon within the frame of the part may be sufficient (especially if your part will be followed by a subsequent part.)) |
| - | Then take the reverse complement of your reverse primer. | + | #Be sure and follow the guidelines above for design of the primer sequence that is complementary to the template. |
| - | + | #Then take the reverse complement of your reverse primer. | |
| - | + | ||
| - | + | Once you have obtained your primers, you can follow the usual PCR protocol using a High Fidelity polymerase. Digest and ligate as usual. | |
| - | + | ||
| - | + | Back to [[Team:Northwestern/Protocol|<span style="color:#4B088A;">Protocol</span>]] | |
| + | |||
| + | [[Category:Protocol]] | ||
Latest revision as of 19:21, 16 October 2010
The following protocol is for creating a new biobricks part from a protein coding sequence. Primer Design
- Design a forward primer to your new BioBrick part comprised of the BioBrick protein coding prefix sequence GTT TCT TCG AAT TCG CGG CCG CTT CTA G followed by the first 20-30 nucleotides of the protein coding region, beginning with the ATG
- Design a reverse primer to your new BioBrick part comprised of the last 20-30 or so nucleotides of the protein coding sequence, excluding the stop codon, followed by the BioBrick protein coding suffix sequence 5'-TAA TAA TAC TAG TAG CGG CCG CTG CAG GAA GAA AC-3'. Note that the BioBrick protein coding suffix sequence includes the double stop codon TAA TAA. (Note: In some cases, for example if you wish use your part directly for making protein fusions using BioScaffold parts [1] or make your part more versatile by giving other users the capacity to do so in the future, then you should only include one stop codon TAA or make a version of your part that has only one stop codon. In this case your sequence should be followed by the BioScaffold and BioBrick compatible protein suffix 5'-TAA TAC TAG TAG CGG CCG CTG CAG GAA GAA AC-3' instead of the BioBrick protein coding suffix sequnce. Since the scar region that is created by the composition of parts, also introduces a stop codon after protein parts, including only one stop codon within the frame of the part may be sufficient (especially if your part will be followed by a subsequent part.))
- Be sure and follow the guidelines above for design of the primer sequence that is complementary to the template.
- Then take the reverse complement of your reverse primer.
Once you have obtained your primers, you can follow the usual PCR protocol using a High Fidelity polymerase. Digest and ligate as usual.
Back to Protocol
