Team:TMU-Tokyo/Notebook experiment/3rd

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Experiment > 3rd week



September 15th
PCR result

1: Fragment 1, 2: Fragment 2, 3: Fragment 3 - Ap,
4: Fragment 3- Cm, 5: pBAD-fimE


Ligation fragment and vector.
Plasmid nameFragment 1-A+BFragment 2Fragment 3 - Ap
FragmentFragment 1-A
Fragment 1-B
Fragment 2Fragment 3 - Ap
VectorpSB1C3pSB1C3pSB1C3


Transformation following plasmid to Escherichia coli MG1655 red.
Fragment 1, 2, 3 - Ap


September 16th
Transformation result.



Mini prep following plasmid.
Fragment 1, 2, 3 - Ap, 3 - Cm, Km-Blunt
Result

1: Fragment 1, 2: Fragment 3 - Ap,
3-5: Fragment 3 - Cm, 6-8: Km-Blunt


PCR following fragment.
FragmentFragment 1Fragment 2Fragment 3 - ApFragment 3 - Cm
PrimerlacA-IRR/IRL
cynR-SaApR
araCHC
araA-TcC
lacI-R(FRT)2
mhpA-SaApR
p(SaApR)-lacIN
mhpA-CmC6
TempleteIF2
FS2
Fragment 2-C
Fragment 2-D
pSG76A2lacI
FRT-Cm2


September 17th
PCR result.

1-2: Fragment 1, 3-4: Fragment 2,
5-6: Fragment 3 - Cm, 8-10: Fragment 3 - Ap


Electro Poration
Fragment 1, 2, 3 - AP to Escherichia coli MG1655 red.


PCR following fragment.
FragmentFragment 1Fragment 2Fragment 3 - ApFragment 3 - Cm
PrimerlacA-IRR/IRL
cynR-SaApR
araCHC
araA-TcC
lacI-R(FRT)2
mhpA-SaApR
p(SaApR)-lacIN
mhpA-CmC6
TempleteIF2
FS2
Fragment 2-C
Fragment 2-D
pSG76A2lacI
FRT-Cm2


September 18th
PCR result.

1-3: Fragment 1, 4-6: Fragment 2,
7-9: Fragment 3 - Ap, 1-3: Fragment 3 - Cm


Mini prep following plasmid
pSB1C3-Km, pSB1A3


PCR following fragment.
FragmentfimEFragment 1Fragment 2Fragment 3 - Cm
PrimerpBAD-N9
fimE-C-EcoRⅠ
lacA-IRR/IRL
cynR-SaApR
araCHC
araA-TcC
R(SaApR)-lacIN
mhpA-CmC6
TempletepBAD
fimE
IF2
FS2
Fragment 2-C
Fragment 2-D
lacI
FRT-Cm


September 19th
PCR result.

1: fimE, 2-3: Fragment 1, 4-5: Fragment 2,
6-7: Fragment 3 - Cm


PCR following fragment. (2nd PCR)
FragmentfimE(1N2)fimE(2N2)fimE(1N9)fimE(2N9)
PrimerpBAD-N2
fimE-1
pBAD-N2
fimE-2
pBAD-N9
fimE-1
pBAD-N9
fimE-2
TempletepBAD
fimE
pBAD
fimE
pBAD
fimE
pBAD
fimE
Result.

1-3: 1N2, 4-6: 2N2, 7-9: 1N9, 10-12: 2N9


Electro Poration
Fragment 1, 2, 3 - Ap to Escherichia coli MG1655 red.


Mini prep following plasmid.
pSB1A3, pSB1C3
Result

1: pSB1A3, 2: pSB1C3


Digestion
fimE-pBAD, pSB1C3-Km, pSB1A3
Result

1: fimE, 2: pSB1C2-Km, 3: pSB1A3


September 20th
Ligation following vector and fragment.
fimE-pBAD + pSB1A3



Check PCR.
Fragment(control)Fragment 1Fragment 2Fragment 3 - Ap
Primer3 sets of the others141-64-1
FLP-R
117-4
pBAD-C3
141-1
SaApR
TempleteEscherichia coli
MG1655 red
(Cell suspention)
(Cell suspention)(Cell suspention)(Cell suspention)
Result

1-5: Fragment 1, 6: control(fragment 1),
7-10: Fragment 2, 11: control(fragment 2),
12-15: Fragment 3, 16: control(fragment 3)


September 21th
Mini prep following plasmid.
Km-Blunt in pSB1C3
Result.

1-3: Km-Blunt in pSB1C3


PCR following fragment.
FragmentFragment 1
PrimerlacA-IRR/IRL
cynR-SaApR
TempleteIF2
FS2
Result.

1-2: Fragment 1


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