From 2007.igem.org

Primer Design

SO1415

Gene sequence: overhang of the actual gene

This sequence was found on http://www.ncbi.nlm.nih.gov/

Megablast was used to compare the designed primer against the s. oneidensis genome

The length, gc content, melt temp etc info was found on www.idtdna.com

The site used to find parameters of a well-designed primer: http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html

SO_1415 gene sequence: 5’ GAATGAATAA ATGAAATGTCCTTCGGACTCCCTGTCCATTTTACGTTGTAATCAAATATTGGATGCGGCTGAAAAGCTCA TTGAGTCACAAGGTGTTGTATCTTTTAAGTTTTCTCAGCTTGCGCATGAGGTGGGATGCTCTACGGGTAC TTTATATAAATTTTTTGAACGTAAAGAAGATGTGTTGGTTTGTTTATTTTTAAGAAGCGCAACCTCAAAT CACTTACCGATATTTATCCATAAAAATCCAGAGTTAACTGCGCAAGAGAAGGTGCTGTTACCCATTTTAT TTACCTTTGAAACCATTAAGCGCAGTAGTAGCTTTTTTACGCTGCGTTCGGTGTCGGTCAATACCATGGT GTGGAAACTGGCCAGTGACGAAAAAGTGGAGCGGTTTAAAAAACGCATTAATGCTTTTTGGAGTTGGTTT ACAGACTCACTGCATTTAGCTGTTGAAAACGGCGAATTAGTGGCAACACCATTACAAATTAAAGAATTGG TCCAAGGGATAACGTTTTATTTAACAGGGTCTTTGACACAATTTGAAAGTCAATTGATTGCCCCAGAGTT TTTGTCTGATCGCCGTGAAACCTGTTATCGACATTTAGCAAACCTGATGGAGCGATACGAGTGGAAAAAG CCTTTAACTCTTGCGCTGTTTGATTCGTTAGAGGCGAGAACTATTAAGTTTTTTGACCAACATTATCGTG ACCATATGACCTGCGCGGCTTGTAGTGCGCTGTCAAATACCGACACTAAGACATCATCTCCCTGTACTCG TCAGTGTGGTTAG GGCGTCCTGC 3’

SO4157

5’ AAGGAAAACC ATGTCCACCATGCTGCCACTGTATTTAGTCGATGATGATGAAGCGATTCTCGACTCCTTAGGGTTTATGC TCAGGCAATTTGGTTACCAAGTACAAACCTTTAGCAGTGGACGGGATTTTTTAGCCCAATGTCCGTTAAC ACAGGCTGGCTGCGTGATTTTAGATAGCCGAATGCCGGAGATCACCGGCCAAGAAGTGCAGCAAAAACTA CTTGAAACCCAAAGCCCATTGGGAGTTATCTTTCTCACGGGGCACGGTGATTTGCCCATGGCATTAAGCG CCTTTCGTAAGGGTGCATGCGATTTTTTTCAAAAGCCGGTATCTGGCAAAGCCCTAGTACAAGCCATTAA AAAAGCGCATAAAGAAAGCCAAGCCAGCTTTGAGCAACAGAGTCTGCAGCATAAATTTGCCCAACTGACC GACCGTGAACAACAAGTGTTAGCCCATGTGGTTCAAGGTATGACCAACAAGCAGATCTCCGAGGCCATGT ATTTATCCTTAAGAACCATTGAAGTGCACCGCGCTAAGATCATGAAAAAGCTCGAAGTCAGTAATATGGC AGAATTAGTACAGCACTTAGCCCACCTAAATACACTCTTACCGGAGTAA TCCAATAAAC 3’

hlyU

ATGAAAACCA TTAATGACAATAAATATTGTTCAATAAATGGATCATCTCACGTACCTCATCACTTTTCAGTGAGTAGAAT ACAGTTTGCGCTTCTTTGCGTGTGGTCACTAAATTATCTTTGCGCAACCAAGCAAGGTGTTGTGATAGTG CCGATTGACTTAAGCCTAATTTTTTATTCATTTCGCCAACGCACATTTCTCCTTCATTCAATAAATAACA AAGGATAAATAAACGGCGTTCGTTTGCGAGTGCCTTTAATAGCACCACGGCATGATCGGCTCGCTCCTGC ATCAATTCAATATTCAT TACGCACTTT

Primer design to be used with the TOPO TA cloning kit

SO1415:

Forward primer:

CTA ACC ACA CTG ACG AGT ACA GGG

LENGTH:24GC CONTENT:54.2 %MELT TEMP:58.6 ºC

Reverse primer:

ATG AAA TGT CCT TCG GAC TCC CTG

LENGTH:24GC CONTENT:50.0 %MELT TEMP:58.7 ºC

SO4157:

Forward primer:

TTA CTC CGG TAA GAG TGT ATT TAG GTG GG

LENGTH:29GC CONTENT:44.8 %MELT TEMP:59.0 ºC

Reverse Primer:

ATG TCC ACC ATG CTG CCA C

LENGTH:19GC CONTENT:57.9 %MELT TEMP:58.4 ºC

hlyU:

Reverse Primer

ATG AAT ATT GAA TTG ATG CAG GAG CGA G

LENGTH:28GC CONTENT:39.3 %MELT TEMP:57.5 ºC

Forward Primer

TTA ATG ACA ATA AAT ATT GTT CAA TAA ATG GAT CAT CTC ACG

LENGTH:42GC CONTENT:26.2 %MELT TEMP:58.0 ºC

Primer design to be used to amplify genes out of the TOPO plasmid (and then incorporated into the pJQ200 plasmid)

SO1415

Forward:

ATATATATGAATTCCTA ACC ACA CTG ACG AGT ACA GGG

LENGTH:24GC CONTENT:54.2 %MELT TEMP:58.6 ºC

Reverse:

AATTAATTAAGCTTATG AAA TGT CCT TCG GAC TCC CTG

LENGTH:24GC CONTENT:50.0 %MELT TEMP:58.7 ºC

SO4157:

Forward:

ATATATATGAATTCTTA CTC CGG TAA GAG TGT ATT TAG GTG GG

LENGTH:29GC CONTENT:44.8 %MELT TEMP:59.0 ºC

Reverse:

AATTAATTAAGCTTATG TCC ACC ATG CTG CCA C

LENGTH:19GC CONTENT:57.9 %MELT TEMP:58.4 ºC

hlyU:

Forward:

ATATATATGAATTCATG AAT ATT GAA TTG ATG CAG GAG CGA G

LENGTH:28GC CONTENT:39.3 %MELT TEMP:57.5 ºC

Reverse:

AATTAATTAAGCTTTTA ATG ACA ATA AAT ATT GTT CAA TAA ATG GAT CAT CTC ACG

LENGTH:42GC CONTENT:26.2 %MELT TEMP:58.0 ºC

(DS)

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