Editing Lambda Red Protocol (section)

== Notes ==

'''Designing necessary primers'''

*First look at the sequence of the plasmid containing the resistance marker you wish to swap in for your target gene.
**[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=15554329 NCBI sequence viewer: pKD3]
***priming site 1: {{blue|GTGTAGGCTGGAGCTGCTTC}}<span style="color: rgb(0, 0, 255); font-family: sans-serif; font-size: 12.7px;">GTGTAGGCTGGAGCTGCTTC</span>
***priming site 2: {{purple|GGACCATGGCTAATTCCCAT}}<span style="color: rgb(128, 0, 128); font-family: sans-serif; font-size: 12.7px;">GGACCATGGCTAATTCCCAT</span>
***priming site 2 reverse complement: {{green|ATGGGAATTAGCCATGGTCC}}<span style="color: rgb(0, 128, 0); font-family: sans-serif; font-size: 12.7px;">ATGGGAATTAGCCATGGTCC</span>



*pKD3 Cat sequence (1034 bases)

<span style="color: rgb(0, 0, 255); font-family: Courier, monospace; font-size: 12.7px;">GTGTAGGCTGGAGCTGCTTC</span><span style="font-family:Courier,monospace;">{{blue|GTGTAGGCTGGAGCTGCTTC}}GAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTTCATTTAAATGGCGCGCCTTACGCCCCGCCC TGCCACTCATCGCAGTACTGTTGTATTCATTAAGCATCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTC GCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGA GACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTC GTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGC CATACGTAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAAT ATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGT GATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGACAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCT TACGTGCCGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCAC AGGTAGGCGCGCCGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTAAGGAGGATATTCATAT{{purple|GGACCATGGCTAATTCCCAT}}</span><span style="color: rgb(128, 0, 128); font-family: Courier, monospace; font-size: 12.7px;">GGACCATGGCTAATTCCCAT</span>

*<span style="font-family:Courier,monospace;">Next, find the sequence (and context sequence) of the gene you wish to remove </span>
**<span style="font-family:Courier,monospace;">In this example, I found the sequence and context for endA (808 bases total) </span>
***<span style="font-family:Courier,monospace;">MG1655_m56_ABE-0009661 +50bp upstream +50bp downstream</span>

<span style="font-family:Courier,monospace;"><span style="font-family:Courier,monospace;">{{orange|
CCAAAACAGCTTTCGCTACGTTGCTGGCTCGTTTTAACACGGAGTAAGTG}}</span></span><span style="color: rgb(255, 165, 0); font-family: Courier, monospace; font-size: 12.7px;">CCAAAACAGCTTTCGCTACGTTGCTGGCTCGTTTTAACACGGAGTAAGTG</span><span style="font-family:Courier,monospace;"><span style="font-family:Courier,monospace;">ATGTACCGTTATTTGTCTATTGCTGCGGTGGTACTGAGCGCAGCATTTTCCGGC CCGGCGTTGGCCGAAGGTATCAATAGTTTTTCTCAGGCGAAAGCCGCGGCGGTAAAAGTCCACGCTGACGCGCCCGGTACGTTTTATTGCGGATGTAAAATTAA CTGGCAGGGCAAAAAAGGCGTTGTTGATCTGCAATCGTGCGGCTATCAGGTGCGCAAAAATGAAAACCGCGCCAGCCGCGTAGAGTGGGAACATGTCGTTCCCG CCTGGCAGTTCGGTCACCAGCGCCAGTGCTGGCAGGACGGTGGACGTAAAAACTGCGCTAAAGATCCGGTCTATCGCAAGATGGAAAGCGATATGCATAACCTG CAGCCGTCAGTCGGTGAGGTGAATGGCGATCGCGGCAACTTTATGTACAGCCAGTGGAATGGCGGTGAAGGCCAGTACGGTCAATGCGCCATGAAGGTCGATTT CAAAGAAAAAGCTGCCGAACCACCAGCGCGTGCACGCGGTGCCATTGCGCGCACCTACTTCTATATGCGCGACCAATACAACCTGACACTCTCTCGCCAGCAAA CGCAGCTGTTCAACGCATGGAACAAGATGTATCCGGTTACCGACTGGGAGTGCGAGCGCGATGAACGCATCGCGAAGGTGCAGGGCAATCATAACCCGTATGTG</span></span>

<span style="font-family:Courier,monospace;"><span style="font-family:Courier,monospace;">CAACGCGCTTGCCAGGCGCGAAAGAGCTAA{{red|CCTACACTAGCGGGATTCTTTTTGTTAACCCCTACCCCACGCGTACAACC}}</span></span><span style="color: rgb(255, 0, 0); font-family: Courier, monospace; font-size: 12.7px;">CCTACACTAGCGGGATTCTTTTTGTTAACCCCTACCCCACGCGTACAACC</span>

*<span style="font-family:Courier,monospace;">Construct primers that have internal overlap with the resistance marker (pKD3) and external overlap with the target knockout gene (endA). </span>
**<span style="font-family:Courier,monospace;">Forward primer: ''A'' </span>
***<span style="font-family:Courier,monospace;">{{orange|CCAAAACAGCTTTCGCTACGTTGCTGGCTCGTTTTAACACGGAGTAAGTG}}{{blue|GTGTAGGCTGGAGCTGCTTC}}</span><span style="font-family: sans-serif; font-size: 12.7px; color: orange;">CCAAAACAGCTTTCGCTACGTTGCTGGCTCGTTTTAACACGGAGTAAGTG</span><span style="font-family: sans-serif; font-size: 12.7px; color: blue;">GTGTAGGCTGGAGCTGCTTC</span>
**<span style="font-family:Courier,monospace;">Reverse primer: ''B'' </span>
***<span style="font-family:Courier,monospace;">{{red|GGTTGTACGCGTGGGGTAGGGGTTAACAAAAAGAATCCCGCTAGTGTAGG}}{{green|ATGGGAATTAGCCATGGTCC}}</span><span style="font-family: sans-serif; font-size: 12.7px; color: red;">GGTTGTACGCGTGGGGTAGGGGTTAACAAAAAGAATCCCGCTAGTGTAGG</span><span style="font-family: sans-serif; font-size: 12.7px; color: green;">ATGGGAATTAGCCATGGTCC</span>

*<span style="font-family:Courier,monospace;">Construct primers that only flank the target gene (endA) for PCR verification </span>
**<span style="font-family:Courier,monospace;">Forward primer: ''C'' </span>
***<span style="font-family:Courier,monospace;">{{orange|CCAAAACAGCTTTCGCTACGTTGCT}} </span><span style="color: rgb(255, 165, 0); font-family: sans-serif; font-size: 12.7px;">CCAAAACAGCTTTCGCTACGTTGCT</span><span style="font-family:Courier,monospace;">(25 bases)</span>
**<span style="font-family:Courier,monospace;">Reverse primer: ''D'' </span>
***<span style="font-family:Courier,monospace;">{{red|GGTTGTACGCGTGGGGTAGGGGTTA}} </span><span style="color: rgb(255, 0, 0); font-family: sans-serif; font-size: 12.7px;">GGTTGTACGCGTGGGGTAGGGGTTA</span><span style="font-family:Courier,monospace;">(25 bases)</span>

*<span style="font-family:Courier,monospace;">Figure out the sequence and size of what you should expect if everything works. In this case, it's Cat inserted into endA flanking region'''(1132 bases total)'''</span>

<span style="font-family:Courier,monospace;"><span style="font-family:Courier,monospace;">{{orange|CCAAAACAGCTTTCGCTACGTTGCTGGCTCGTTTTAACACGGAGTAAGTG}}{{blue|GTGTAGGCTGGAGCTGCTTC}}</span></span><span style="font-family: Courier, monospace; font-size: 12.7px; color: orange;">CCAAAACAGCTTTCGCTACGTTGCTGGCTCGTTTTAACACGGAGTAAGTG</span><span style="font-family: Courier, monospace; font-size: 12.7px; color: blue;">GTGTAGGCTGGAGCTGCTTC</span><span style="font-family:Courier,monospace;"><span style="font-family:Courier,monospace;">GAAGTTCCTATACTTTCTAGAGAATAGGAACTTC GGAATAGGAACTTCATTTAAATGGCGCGCCTTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTATTCATTAAGCATCTGCCGCATGGAAGCCATCACAAA CGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCC ACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACA CGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAAC AAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGTAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGA TAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAA ATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGACAACTCAAAAA ATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTA TCAACAGGGACACCAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCACAGGTAGGCGCGCCGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAG GAACTAAGGAGGATATTCATAT{{purple|GGACCATGGCTAATTCCCAT}}{{red|CCTACACTAGCGGGATTCTTTTTGTTAACCCCTACCCCACGCGTACAACC}}</span></span><span style="color: rgb(255, 0, 0); font-family: Courier, monospace; font-size: 12.7px;">CCTACACTAGCGGGATTCTTTTTGTTAACCCCTACCCCACGCGTACAACC</span>