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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’

Primer 1:

TGA ATA AAT GAA ATG TCC TTC GGA CTC CCT G

LENGTH:31

GC CONTENT:41.9 %

MELT TEMP:59.9 ºC

MOLECULAR WEIGHT:9494.2 g/mole

EXTINCTION COEFFICIENT:298500 L/(mole·cm)

nmole/OD260:3.35

µg/OD260:31.81

Primer 2:

GAC GCC CTA ACC ACA CTG ACG

LENGTH:21

GC CONTENT:61.9 %

MELT TEMP:60.2 ºC

MOLECULAR WEIGHT:6345.2 g/mole

EXTINCTION COEFFICIENT:197000 L/(mole·cm)

nmole/OD260:5.08

µg/OD260:32.21

SO4157

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

Primer 1:

AAA ACC ATG TCC ACC ATG CTG C

LENGTH:22

GC CONTENT:50.0 %

MELT TEMP:58.7 ºC

MOLECULAR WEIGHT:6648.4 g/mole

EXTINCTION COEFFICIENT:207700 L/(mole·cm)

nmole/OD260:4.81

µg/OD260:32.01

Primer 2: ATT GGA TTA CTC CGG TAA GAG TGT ATT TAG GT

LENGTH:32

GC CONTENT:37.5 %

MELT TEMP:58.6 ºC

MOLECULAR WEIGHT:9924.5 g/mole

EXTINCTION COEFFICIENT:320800 L/(mole·cm)

nmole/OD260:3.12

µg/OD260:30.94

hlyU

ATGAAAACCA TTAATGACAATAAATATTGTTCAATAAATGGATCATCTCACGTACCTCATCACTTTTCAGTGAGTAGAAT ACAGTTTGCGCTTCTTTGCGTGTGGTCACTAAATTATCTTTGCGCAACCAAGCAAGGTGTTGTGATAGTG CCGATTGACTTAAGCCTAATTTTTTATTCATTTCGCCAACGCACATTTCTCCTTCATTCAATAAATAACA AAGGATAAATAAACGGCGTTCGTTTGCGAGTGCCTTTAATAGCACCACGGCATGATCGGCTCGCTCCTGC ATCAATTCAATATTCAT TACGCACTTT

Primer 1: ATG AAA ACC ATT AAT GAC AAT AAA TAT TGT TCA ATA AAT GG

LENGTH:41

GC CONTENT:22.0 %

MELT TEMP:56.6 ºC

MOLECULAR WEIGHT:12655.3 g/mole

EXTINCTION COEFFICIENT:432600 L/(mole·cm)

nmole/OD260:2.31

µg/OD260:29.25

Primer 2: GTG CGT AAT GAA TAT TGA ATT GAT GCA GGA

LENGTH:30

GC CONTENT:36.7 %

MELT TEMP:57.8 ºC

MOLECULAR WEIGHT:9349.1 g/mole

EXTINCTION COEFFICIENT:309700 L/(mole·cm)

nmole/OD260:3.23

µg/OD260:30.19