Difference between revisions of "Primer Design for the eCDM8 and RFP"
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PCR primers for insertion of Genes between Junctions C and D | PCR primers for insertion of Genes between Junctions C and D | ||
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1. Conduct Inverse PCR using scaffold with promoters and C dogs and J-GGA_C_rev and J-GGA_D_for. | 1. Conduct Inverse PCR using scaffold with promoters and C dogs and J-GGA_C_rev and J-GGA_D_for. | ||
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3. Conduct J-GGA with products from 1 and 2 above. | 3. Conduct J-GGA with products from 1 and 2 above. | ||
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Gene primer considerations. | Gene primer considerations. | ||
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5. 18 nt of gene coding sequence on each primer | 5. 18 nt of gene coding sequence on each primer | ||
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Primer designs for the eCDM8 gene | Primer designs for the eCDM8 gene | ||
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Forward ECDM8 gcatggtctcacatcaaagaaatgccgcagccg | Forward ECDM8 gcatggtctcacatcaaagaaatgccgcagccg | ||
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>BBa_J100100 Part-only sequence (3204 bp) | >BBa_J100100 Part-only sequence (3204 bp) | ||
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gaattcgcggccgcttctagagggtctcgaatgaccatcaaagaaatgccgcagccgaaaacctttggtgaactgaaaaatctgccgctgctgaataccg | gaattcgcggccgcttctagagggtctcgaatgaccatcaaagaaatgccgcagccgaaaacctttggtgaactgaaaaatctgccgctgctgaataccg | ||
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Primer designs for the RFP gene | Primer designs for the RFP gene | ||
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RFP Forward gcatggtctcgcatcgcttcctccgaagacgtt | RFP Forward gcatggtctcgcatcgcttcctccgaagacgtt | ||
RFP Rev ccagggtctcatccgttattaagcaccggtgga | RFP Rev ccagggtctcatccgttattaagcaccggtgga | ||
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5’ Tccaccggtgcttaataa 3’ | 5’ Tccaccggtgcttaataa 3’ | ||
3’ Aggtggccacgaattatt 5’ | 3’ Aggtggccacgaattatt 5’ | ||
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RFP Gene Sequence | RFP Gene Sequence | ||
Latest revision as of 15:51, 11 June 2013
PCR primers for insertion of Genes between Junctions C and D
1. Conduct Inverse PCR using scaffold with promoters and C dogs and J-GGA_C_rev and J-GGA_D_for.
2. Conduct PCR of eCDM8 gene and RFP gene templates with primers below.
3. Conduct J-GGA with products from 1 and 2 above.
Gene primer considerations.
1. Four extra bases on 5’ end (eg. GCAT)
2. BsaI site directed toward the gene
3. Sticky end of 5’ CATC 3’ for Junction C on For primer
4. Sticky end of 5’ TCCG 3’ for Junction D on Rev primer
5. 18 nt of gene coding sequence on each primer
Primer designs for the eCDM8 gene
Forward ECDM8 gcatggtctcacatcaaagaaatgccgcagccg
Rev ECDM8 ccagggtctcatccgttattaacctgcccaaac
5’ gttTgggcaggttaataa 3’
3’ caaacccgtccaattatt 5’
>BBa_J100100 Part-only sequence (3204 bp)
gaattcgcggccgcttctagagggtctcgaatgaccatcaaagaaatgccgcagccgaaaacctttggtgaactgaaaaatctgccgctgctgaataccg
ataaaccggttcatgcactgatgaaaattgcagatgaactgggcgagatctttaaattcgaagcaccgggtagcgttacccgttatctgagcagccagcg
tctgattaaagaagcatgcgacgaaagccgctttgataaaaatctgattcagtggctgaaattcatccgcgattttctgggtgatggtctgagcaccagc
tggacccatgaaaaaaactggaaaaaagcccataatattctgctgccgagctttagccagcaggcaatgaaaggttatcatgcaatgatggttgatattg
ccgttcagctggttcagaaatgggaacgtctgaatgcagatgaacatattgaagttccggaagatatgacccgtctgaccctggataccattggtctgtg
tggttttaactatcgcttcaacagcttttatcgtgatcagccgcatccgtttgttaccagcatggttcgtgcactggatgaagcagtgaataaatggcag
cgtgcaaatccggatgatccggcatatgatgaaaataaacgtcagttccaagaggatattaaagtgatgaatgacctggtggacaaaattatcgcagatc
gtaaagcaagcggtgaacagagtgatgatctgctgacccatatgctgaatggtaaagatccggaaaccggtgaaccgctggatgatgaaaacattcgtta
tcagattattaccttcctgattgccggtcatgaaaccaccagtggtctgctgagctttgcactgtattttctggttaaaaatccgcacgttctgcaaaaa
gcagccgaagaagcagcacgcgttctggttgatccggttccgagctataaacaggttaaacagctgaaatatgtgggcatggttctgaatgaagcactgc
gtctgtggccgacctttccgtggtttagcctgtatgcaaaagaagataccgttctgggaggtgaatatccgctggaaaaaggtgatgaactgatggttct
gattccgcagctgcatcgtgataaaaccatttggggtgatgatgtggaagaatttcgtccggaacgttttgaaaatccgagcgcaattccgcagcatgcc
tttaaaccgtttggtaatggtcagcgtgcatgtattggtcagcagtttgcactgcatgaagccaccctggttctgggtatgatgctgaaacattttgatt
ttgaggaccacaccaactatgagctggatattaaaggcaccctgaccctgaaaccggaaggttttgttgttaaagccaaaagcaaaaaaatcccgctggg
tggtattccgagcccgagcaccgaacagagcgcaaaaaaagttcgtaaaaaagccgaaaacgcccataatacaccgctgctggttctgtatggtagcaat
atgggtacagccgaaggcaccgcacgtgatctggcagatattgcaatgagcaaaggttttgcaccgcaggttgcaaccctggatagccatgcaggtaatc
tgcctcgtgaaggtgcagttctgattgttaccgcaagctataatggtcatccgcctgataatgcaaaacagtttgttgattggctggatcaggcaagcgc
agatgaagttaaaggtgttcgttatagcgtttttggttgcggtgataaaaactgggcaaccacctatcagaaagttccggcatttattgatgaaaccctg
gcagcaaaaggtgcagaaaatattgcagatcgtggtgaaaccgatgccagtgatgattttgaaggcacctatgaagaatggcgtgaacatatgtggtcag
atgttgcagcctattttaacctggatatcgaaaacagcgaggacaataaaagcaccctgagcctgcaatttgtggatagcgcagcagatatgccgctggc
aaaaatgcatggtgcatttagcaccaatgttgttgcaagcaaagaactgcaacagcctggtagcgcacgtagcacccgtcatctggaaattgaactgccg
aaagaagcaagctatcaagagggtgatcatctgggtgttattccgcgtaattatgaaggtattgttaatcgtgttaccgcacgttttggtctggatgcaa
gccagcagattcgtctggaagcagaagaagaaaaactggcacatctgccgctggccaaaaccgttagcgttgaagaactgctgcaatatgttgaactgca
agatccggttacccgtacccagctgcgtgcaatggcagcaaaaaccgtttgtccgcctcataaagttgaactggaagcactgctggagaaacaggcatat
aaagaacaggttctggcaaaacgtctgaccatgctggaactgctggaaaaatatccggcatgcgaaatgaaattcagcgaatttattgcactgctgccga
gtattcgtccgcgttattatagcattagcagcagtccgcgtgttgatgaaaaacaggccagcattaccgttagcgtggttagcggtgaagcatggtcagg
ttatggtgaatataaaggtattgccagcaattatctggccgaactgcaagaaggtgataccattacctgttttattagcacaccgcagagcgaatttacc
ctgccgaaagatcctgaaacaccgctgattatggttggtccgggtacaggtgttgcaccgtttcgtggttttgttcaggcacgtaaacaactgaaagaac
agggtcagagcctgggtgaagcacatctgtattttggttgtcgtagtccgcatgaggattatctgtatcaagaagaactggaaaacgcacagagcgaagg
tattattaccctgcataccgcatttagccgtatgccgaatcagccgaaaacatatgttcagcatgttatggaacaggatggcaaaaaactgattgaactg
ctggatcagggtgcccatttctatatttgtggtgatggtagccagatggcaccggcagttgaagcaaccctgatgaaaagctatgcagatgttcatcagg
ttagcgaagcagatgcacgtctgtggctgcaacagctggaagaaaaaggtcgttatgcaaaagatgtttgggcaggttaataatactagtagcggccgct gcag
Primer designs for the RFP gene
RFP Forward gcatggtctcgcatcgcttcctccgaagacgtt
RFP Rev ccagggtctcatccgttattaagcaccggtgga
5’ Tccaccggtgcttaataa 3’
3’ Aggtggccacgaattatt 5’
RFP Gene Sequence
>BBa_E1010 Part-only sequence (706 bp)
atggcttcctccgaagacgttatcaaagagttcatgcgtttcaaagttcgtatggaaggttccgttaacggtcacgagttcgaaatcgaaggtgaaggtg
aaggtcgtccgtacgaaggtacccagaccgctaaactgaaagttaccaaaggtggtccgctgccgttcgcttgggacatcctgtccccgcagttccagta
cggttccaaagcttacgttaaacacccggctgacatcccggactacctgaaactgtccttcccggaaggtttcaaatgggaacgtgttatgaacttcgaa
gacggtggtgttgttaccgttacccaggactcctccctgcaagacggtgagttcatctacaaagttaaactgcgtggtaccaacttcccgtccgacggtc
cggttatgcagaaaaaaaccatgggttgggaagcttccaccgaacgtatgtacccggaagacggtgctctgaaaggtgaaatcaaaatgcgtctgaaact
gaaagacggtggtcactacgacgctgaagttaaaaccacctacatggctaaaaaaccggttcagctgccgggtgcttacaaaaccgacatcaaactggac
atcacctcccacaacgaagactacaccatcgttgaacagtacgaacgtgctgaaggtcgtcactccaccggtgcttaataa
