PHO80cds

From 2007.igem.org

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GenePho
GenePho
-
CODINGSEQUENCE
+
CODING SEQUENCE
ATGGAAAGCACATCAGGAGAACGTTCCGAAAATATACATG
ATGGAAAGCACATCAGGAGAACGTTCCGAAAATATACATG
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Pho cds
Pho cds
-
882bp e 293aa
+
882bp 293aa
1)91 bp position 31-32aa SR
1)91 bp position 31-32aa SR
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Frame 5’-3’
Frame 5’-3’
VirtualSequence:
VirtualSequence:
 +
MESTSGERSENIHEDQGIPKVILPADFNKC[[SR]]TDLVVLISRMLVSLIAINENSATKKSDD
MESTSGERSENIHEDQGIPKVILPADFNKC[[SR]]TDLVVLISRMLVSLIAINENSATKKSDD
QITLTRYHSKIPPNISIFNYFIRLTKFSSLEHCVLMTSLYYIDLLQTVYPDFTLNSLTAH
QITLTRYHSKIPPNISIFNYFIRLTKFSSLEHCVLMTSLYYIDLLQTVYPDFTLNSLTAH
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t648c antisense 5'-ctattcttcgatagtatttatctaggacattatatccactttttggacgat-3'
t648c antisense 5'-ctattcttcgatagtatttatctaggacattatatccactttttggacgat-3'
 +
 +
'''
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results of pho80 coding sequence'''
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 +
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''PCR of mutagenesis of pho 80 from topo TA plasmid''
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Finally volume is 50μl
 +
{| border="1"
 +
|-
 +
! Acqua, µl
 +
| 40
 +
|-
 +
! Buffer, µl
 +
| 5
 +
|-
 +
! dNTPs, µl
 +
| 1
 +
|-
 +
! Taq (Pfu turbo), µl
 +
| 1
 +
|-
 +
! PrimerFw (125 ng), µl
 +
| 1
 +
|-
 +
! PrimerRev (125 ng), µl
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| 1
 +
|-
 +
! Template (5 ng), µl
 +
| 1
 +
|}
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 +
Cycling Conditions
 +
 +
 +
{| border="1"
 +
|-
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! I°CYCLE
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| 95°c for 30 seconds
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|-
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! 12 CYCLES
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| denaturation  95°C for 30 seconds
 +
| annealing    55°c for 1 minutes
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| elongation    68°c for 5 minutes
 +
|-
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! LAST CYCLE
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| 72°C for 2 minutes
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| 4°c
 +
|}
 +
 +
 +
PRIMERS
 +
 +
PHO 80 fw Xba I
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5'- GC TCTAGA ATGGAAAGCACATCAGGAG-3'
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Tm 65.1°C
 +
 +
PHO 80 rew Spe I
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5'-GG ACTAGT ATCTGGCTTTGATCGCTTG-3'
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Tm 65.1°C
 +
 +
[[PCR]] of pho80 from DNA yeast
 +
 +
[[Image:geldoc pho 1.jpg]]
 +
 +
 +
midi pho80 in topo TA plasmid
 +
 +
[[Image:geldoc pho 2.jpg]]
 +
 +
 +
digestion with Dpn I after first mutation of pho 80
 +
 +
[[Image:geldoc pho 3.jpg]]
 +
 +
 +
digestion with Dpn I after second mutation of pho 80
 +
 +
[[Image:geldoc pho 4.jpg]]
 +
 +
 +
midi pho 80 mutated
 +
 +
[[Image:geldoc pho 5.jpg]]
 +
 +
 +
digestion pho 80 mutated with Spe i and Xba I
 +
 +
[[Image:geldoc pho 6.jpg]]

Latest revision as of 16:53, 25 October 2007

GenePho CODING SEQUENCE

ATGGAAAGCACATCAGGAGAACGTTCCGAAAATATACATG AGGATCAAGGGATACCAAAAGTAATTCTGCCCGCTGATTT TAATAAATGCTCTAGAACTGACCTAGTGGTGCTCATATCA CGAATGTTAGTATCGCTGATAGCAATCAATGAAAATTCAG CAACAAAGAAATCTGATGACCAAATTACTTTAACACGATA CCATTCTAAGATTCCTCCAAACATATCAATCTTCAACTAT TTCATACGACTGACAAAGTTTTCCTCTTTAGAACATTGTG TGCTTATGACATCACTCTATTATATCGATTTATTGCAAAC TGTGTATCCTGATTTTACGCTTAATTCGTTGACTGCCCAT AGGTTTTTATTAACAGCCACCACAGTCGCAACAAAAGGCT TATGTGATTCGTTCTCAACAAACGCCCATTATGCAAAAGT TGGAGGAGTACGATGTCACGAATTGAATATACTGGAGAAC GATTTTTTAAAGAGAGTAAACTACAGAATCATTCCGCGGG ATCATAACATTACGTTATGTAGTATAGAGCAAAAACAGAA AAAGTTTGTCATAGATAAAAACGCATTAGGGTCTCTCGAT TTGGATTCTTATTCTTACGTTAATCGTCCAAAAAGTGGAT ATAATGTTCTAGATAAATACTATCGAAGAATAGTTCAGCT GGTGGGTTCCTTTAACGCTTCACCTGATAAGAGTAGAAAG GTTGATTATGTTCTCCCGCCAAATATTGATATAGTGAGTG AAAGTGGCTCCCAAACTACTCAACTAAAGGGGTCGTCATC ACCCAATTCTCACTCTTCACAAAAGCGATATTCTGAGGCA AAGGACGCACATATCTATAACAAGCGATCAAAGCCAGATT AA

XbaI restriction sequence TCTAGA cuts in sites 91 648


Pho cds 882bp 293aa

1)91 bp position 31-32aa SR S=TCTTCCTCATCG

R=AGAAGG

2)648 bp position 216-217-218aa VLD V=TTGTCGTAGTG

L=TTATTGCTTCTACTCCTG

D=GATGAC


Frame 5’-3’ VirtualSequence:

MESTSGERSENIHEDQGIPKVILPADFNKCSRTDLVVLISRMLVSLIAINENSATKKSDD QITLTRYHSKIPPNISIFNYFIRLTKFSSLEHCVLMTSLYYIDLLQTVYPDFTLNSLTAH RFLLTATTVATKGLCDSFSTNAHYAKVGGVRCHELNILENDFLKRVNYRIIPRDHNITLC SIEQKQKKFVIDKNALGSLDLDSYSYVNRPKSGYNVLDKYYRRIVQLVGSFNASPDKSRK VDYVLPPNIDIVSESGSQTTQLKGSSSPNSHSSQKRYSEAKDAHIYNKRSKPD


PHO80 Primer sequences:

t93c 5'-cgctgattttaataaatgctccagaactgacctagtggtgc-3'

t93c antisense 5'-gcaccactaggtcagttctggagcatttattaaaatcagcg-3'

t648c 5'-atcgtccaaaaagtggatataatgtcctagataaatactatcgaagaatag-3'

t648c antisense 5'-ctattcttcgatagtatttatctaggacattatatccactttttggacgat-3'

results of pho80 coding sequence


PCR of mutagenesis of pho 80 from topo TA plasmid Finally volume is 50μl

Acqua, µl 40
Buffer, µl 5
dNTPs, µl 1
Taq (Pfu turbo), µl 1
PrimerFw (125 ng), µl 1
PrimerRev (125 ng), µl 1
Template (5 ng), µl 1

Cycling Conditions


I°CYCLE 95°c for 30 seconds
12 CYCLES denaturation 95°C for 30 seconds annealing 55°c for 1 minutes elongation 68°c for 5 minutes
LAST CYCLE 72°C for 2 minutes 4°c


PRIMERS

PHO 80 fw Xba I 5'- GC TCTAGA ATGGAAAGCACATCAGGAG-3' Tm 65.1°C

PHO 80 rew Spe I 5'-GG ACTAGT ATCTGGCTTTGATCGCTTG-3' Tm 65.1°C

PCR of pho80 from DNA yeast

Geldoc pho 1.jpg


midi pho80 in topo TA plasmid

Geldoc pho 2.jpg


digestion with Dpn I after first mutation of pho 80

Geldoc pho 3.jpg


digestion with Dpn I after second mutation of pho 80

Geldoc pho 4.jpg


midi pho 80 mutated

Geldoc pho 5.jpg


digestion pho 80 mutated with Spe i and Xba I

Geldoc pho 6.jpg

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