Boston UniversityPrimerDesign

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[https://2007.igem.org/Boston_UniversityStatus Back]
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== Primer Design ==
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SO1415
SO1415
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3’
3’
-
Primer 1:
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SO4157
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'''TGA ATA A'''AT GAA ATG TCC TTC GGA CTC CCT G
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5’
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'''AAGGAAAACC'''
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ATGTCCACCATGCTGCCACTGTATTTAGTCGATGATGATGAAGCGATTCTCGACTCCTTAGGGTTTATGC
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TCAGGCAATTTGGTTACCAAGTACAAACCTTTAGCAGTGGACGGGATTTTTTAGCCCAATGTCCGTTAAC
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ACAGGCTGGCTGCGTGATTTTAGATAGCCGAATGCCGGAGATCACCGGCCAAGAAGTGCAGCAAAAACTA
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CTTGAAACCCAAAGCCCATTGGGAGTTATCTTTCTCACGGGGCACGGTGATTTGCCCATGGCATTAAGCG
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CCTTTCGTAAGGGTGCATGCGATTTTTTTCAAAAGCCGGTATCTGGCAAAGCCCTAGTACAAGCCATTAA
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AAAAGCGCATAAAGAAAGCCAAGCCAGCTTTGAGCAACAGAGTCTGCAGCATAAATTTGCCCAACTGACC
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GACCGTGAACAACAAGTGTTAGCCCATGTGGTTCAAGGTATGACCAACAAGCAGATCTCCGAGGCCATGT
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ATTTATCCTTAAGAACCATTGAAGTGCACCGCGCTAAGATCATGAAAAAGCTCGAAGTCAGTAATATGGC
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AGAATTAGTACAGCACTTAGCCCACCTAAATACACTCTTACCGGAGTAA
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'''TCCAATAAAC'''
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3’
-
LENGTH:31
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hlyU
-
GC CONTENT:41.9 %
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'''ATGAAAACCA'''
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TTAATGACAATAAATATTGTTCAATAAATGGATCATCTCACGTACCTCATCACTTTTCAGTGAGTAGAAT
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ACAGTTTGCGCTTCTTTGCGTGTGGTCACTAAATTATCTTTGCGCAACCAAGCAAGGTGTTGTGATAGTG
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CCGATTGACTTAAGCCTAATTTTTTATTCATTTCGCCAACGCACATTTCTCCTTCATTCAATAAATAACA
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AAGGATAAATAAACGGCGTTCGTTTGCGAGTGCCTTTAATAGCACCACGGCATGATCGGCTCGCTCCTGC
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ATCAATTCAATATTCAT
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'''TACGCACTTT'''
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MELT TEMP:59.9 ºC
 
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MOLECULAR WEIGHT:9494.2 g/mole
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'''Primer design to be used with the TOPO TA cloning kit'''
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EXTINCTION COEFFICIENT:298500 L/(mole·cm)
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SO1415:
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nmole/OD260:3.35
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Forward primer:
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µg/OD260:31.81
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CTA ACC ACA CTG ACG AGT ACA GGG
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Primer 2:
+
LENGTH:24GC CONTENT:54.2 %MELT TEMP:58.6 ºC
-
'''GAC GCC''' CTA ACC ACA CTG ACG
 
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LENGTH:21
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Reverse primer:
-
GC CONTENT:61.9 %
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ATG AAA TGT CCT TCG GAC TCC CTG
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MELT TEMP:60.2 ºC  
+
LENGTH:24GC CONTENT:50.0 %MELT TEMP:58.7 ºC
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MOLECULAR WEIGHT:6345.2 g/mole
 
-
EXTINCTION COEFFICIENT:197000 L/(mole·cm)
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SO4157:
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nmole/OD260:5.08
 
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µg/OD260:32.21
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Forward primer:
 +
TTA CTC CGG TAA GAG TGT ATT TAG GTG GG
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LENGTH:29GC CONTENT:44.8 %MELT TEMP:59.0 ºC
-
SO4157
 
-
5’
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Reverse Primer:
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'''AAGGAAAACC'''
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ATGTCCACCATGCTGCCACTGTATTTAGTCGATGATGATGAAGCGATTCTCGACTCCTTAGGGTTTATGC
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-
TCAGGCAATTTGGTTACCAAGTACAAACCTTTAGCAGTGGACGGGATTTTTTAGCCCAATGTCCGTTAAC
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ACAGGCTGGCTGCGTGATTTTAGATAGCCGAATGCCGGAGATCACCGGCCAAGAAGTGCAGCAAAAACTA
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CTTGAAACCCAAAGCCCATTGGGAGTTATCTTTCTCACGGGGCACGGTGATTTGCCCATGGCATTAAGCG
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CCTTTCGTAAGGGTGCATGCGATTTTTTTCAAAAGCCGGTATCTGGCAAAGCCCTAGTACAAGCCATTAA
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AAAAGCGCATAAAGAAAGCCAAGCCAGCTTTGAGCAACAGAGTCTGCAGCATAAATTTGCCCAACTGACC
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GACCGTGAACAACAAGTGTTAGCCCATGTGGTTCAAGGTATGACCAACAAGCAGATCTCCGAGGCCATGT
+
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ATTTATCCTTAAGAACCATTGAAGTGCACCGCGCTAAGATCATGAAAAAGCTCGAAGTCAGTAATATGGC
+
-
AGAATTAGTACAGCACTTAGCCCACCTAAATACACTCTTACCGGAGTAA
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-
'''TCCAATAAAC'''
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-
3’
+
-
Primer 1:
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ATG TCC ACC ATG CTG CCA C
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'''AAA ACC''' ATG TCC ACC ATG CTG C
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LENGTH:19GC CONTENT:57.9 %MELT TEMP:58.4 ºC
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LENGTH:22
 
-
GC CONTENT:50.0 %
+
hlyU:
-
MELT TEMP:58.7 ºC
 
-
MOLECULAR WEIGHT:6648.4 g/mole
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Reverse Primer
-
EXTINCTION COEFFICIENT:207700 L/(mole·cm)
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ATG AAT ATT GAA TTG ATG CAG GAG CGA G
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nmole/OD260:4.81
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LENGTH:28GC CONTENT:39.3 %MELT TEMP:57.5 ºC
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µg/OD260:32.01
 
-
Primer 2:
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Forward Primer
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'''ATT GGA''' TTA CTC CGG TAA GAG TGT ATT TAG GT
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-
LENGTH:32
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TTA ATG ACA ATA AAT ATT GTT CAA TAA ATG GAT CAT CTC ACG
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GC CONTENT:37.5 %
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LENGTH:42GC CONTENT:26.2 %MELT TEMP:58.0 ºC
-
MELT TEMP:58.6 ºC
 
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MOLECULAR WEIGHT:9924.5 g/mole
 
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EXTINCTION COEFFICIENT:320800 L/(mole·cm)
 
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nmole/OD260:3.12
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'''Primer design to be used to amplify genes out of the TOPO plasmid (and then incorporated into the pJQ200 plasmid)'''
-
µg/OD260:30.94
 
 +
SO1415
-
hlyU
 
-
'''ATGAAAACCA'''
+
Forward:
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TTAATGACAATAAATATTGTTCAATAAATGGATCATCTCACGTACCTCATCACTTTTCAGTGAGTAGAAT
+
 
-
ACAGTTTGCGCTTCTTTGCGTGTGGTCACTAAATTATCTTTGCGCAACCAAGCAAGGTGTTGTGATAGTG
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ATATATATGAATTCCTA ACC ACA CTG ACG AGT ACA GGG
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CCGATTGACTTAAGCCTAATTTTTTATTCATTTCGCCAACGCACATTTCTCCTTCATTCAATAAATAACA
+
 
-
AAGGATAAATAAACGGCGTTCGTTTGCGAGTGCCTTTAATAGCACCACGGCATGATCGGCTCGCTCCTGC
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LENGTH:24GC CONTENT:54.2 %MELT TEMP:58.6 ºC
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ATCAATTCAATATTCAT
+
 
-
'''TACGCACTTT'''
+
 
 +
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
 +
 
-
Primer 1:
+
Reverse:
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'''ATG AAA ACC A'''TT AAT GAC AAT AAA TAT TGT TCA ATA AAT GG
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-
LENGTH:41
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AATTAATTAAGCTTATG TCC ACC ATG CTG CCA C
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GC CONTENT:22.0 %
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LENGTH:19GC CONTENT:57.9 %MELT TEMP:58.4 ºC
-
MELT TEMP:56.6 ºC
 
-
MOLECULAR WEIGHT:12655.3 g/mole
 
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EXTINCTION COEFFICIENT:432600 L/(mole·cm)
+
hlyU:
-
nmole/OD260:2.31
 
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µg/OD260:29.25
+
Forward:
-
Primer 2:
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ATATATATGAATTCATG AAT ATT GAA TTG ATG CAG GAG CGA G
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'''GTG CGT A'''AT GAA TAT TGA ATT GAT GCA GGA
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LENGTH:30
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LENGTH:28GC CONTENT:39.3 %MELT TEMP:57.5 ºC
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GC CONTENT:36.7 %
 
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MELT TEMP:57.8 ºC
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Reverse:
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MOLECULAR WEIGHT:9349.1 g/mole
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AATTAATTAAGCTTTTA ATG ACA ATA AAT ATT GTT CAA TAA ATG GAT CAT CTC ACG
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EXTINCTION COEFFICIENT:309700 L/(mole·cm)
+
LENGTH:42GC CONTENT:26.2 %MELT TEMP:58.0 ºC
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nmole/OD260:3.23
 
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µg/OD260:30.19
+
(DS)

Latest revision as of 15:07, 13 June 2007

Back

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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