Boston University/Primer Design
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)
