Team:TMU-Tokyo/Notebook experiment/4th

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Experiment > 4th week


September 22th
Mini prep following plasmid.
fimE-pBAD(in pSB1A3), pSB1C3-Km, pSB1A3-Km
Result.

1: pSB1C3-Km, 2-4: pSB1A3-Km, 5-6: fimE-pBAD


Check PCR.
Fragment(control)Fragment 3 - ApCm
Primer141-49
SaApL
141-49
SaApL
TempleteEscherichia coli
MG1655 red
(Cell suspention)
(Cell suspention)
Result.

1-9: Fragment 3 - ApCm

Check PCR.
Fragment(control)Fragment 3 - ApCmFragment 3 - ApCm
Primer2 sets of the others141-1
141-20
141-1
141-74
TempleteEscherichia coli
MG1655 red
(Cell suspention)
(Cell suspention)
Result.

1-7: 141-1 and 141-20, 8: control(141-1 and 141-20),
9-15: 141-1 and 141-74, 16: control(141-1 and 141-74),


Digestion following plasmid.
pSB1C3-Km-Blunt


P1 Preparation following fragment.
Fragment 1, 2


September 23th
PCR following fragment.
FragmentFragment 3 - ApCm
PrimermhpA-CmC6
SaApL
TempleteFragment 3 - Cm
Ap(SaApR-SaApL)
Result.

1: Fragment 3 - Cm, 2: Fragment 3 - ApCm,
3: pSB1C3-Km-Blunt


Check PCR.
FragmentFragment 3 - Ap
(control)
Fragment 3 - ApCm
PrimerCmNR-Not
SaApL
CmNR-Not
SaApL
Templete(Cell suspention)(Cell suspention)
Result.

1-31: Fragment 3 - ApCm


Electro Pration
Fragment 3 - Cm to Escherichia coli MG1655 red.


P1 Preparation
Fragment 3 - ApCm


September 24th
P1 transdunction
Fragment 1, 2
Result.



FragmentFragment 3 - Ap
(control)
Fragment 3 - ApCm
PrimerCmNR-Not
141-1
CmNR-Not
141-1
Templete(Cell suspention)(Cell suspention)
Result.

1-16: Fragment 3 - ApCm


P1 transduction
Fragment 2 to Fragment 1
Fragment 1 to Fragment 2
Result.


Get Fragment 1-2

Electro Poration
Fragment 1, 2, 3 - Ap, 3 - Cm, 3 - ApCm (in pSBC3-Km) to Escherichia coli DH1
Result.



September 25th
Check PCR.
FragmentFragment 3 - ApCm
(control)
Fragment 3 - ApCm
Primer141-49
141-1
CmNR-Not
141-1
Templete(Cell suspention)(Cell suspention)
Result.

1-7: 141-49 and 141-1, 8: control(141-49 and 141-8),
9-15: 141-1 and CmNR-Not, 16: control(141-1 and CmNR-Not)


September 26th
P1 transduction
Fragment 3 to Fragment 1-2
Get Fragment 1-2-3.


Check PCR
FragmentFragment 1-2-3Fragment 1-2
(control)
PrimerCmNR-Not
141-1
141-49
141-1
Templete(Cell suspention)(Cell suspention)
Result.

1-7: Fragment 1-2-3, 8: cotrol, 9-10: Fragment 1,
11-13: Fragment 2, 14-15: Fragment 3 - Ap, 16-17: Fragment 3 - Cm
18-19: Fragment 3 - AmCm


September 27th
Assay
Pythagorean devices
LB 2ml + arabinose -10% 0.1ml + with the inoculating loop 1655 ins①~③ No.1
LB 2ml + arabinose -10% 0.1ml + with the inoculating loop 1655 ins①~③ No.2
LB 2ml + arabinose -10% 0.1ml + with the inoculating loop 1655 ins①~③ No.3
LB 2ml + arabinose -10% 0.1ml + 1655
LB 2ml + with the inoculating loop 1655 ins①~③ No.1
LB 2ml + with the inoculating loop 1655 ins①~③ No.2
LB 2ml + with the inoculating loop 1655 ins①~③ No.3
LB 2ml + 1655
⇒I culture it at 39 degrees for 2h.

ara(+) No.1~3 ara(-) No.1~3 ara(+) ara(-)
Premix(1) Cell suspension ins①-③
Ins①-③
1655
1655
GXL Buff 20
dNTP 8
GXL 2
(30)
Premix(2) Premix(1) 27
CmNR-not2 9
141-1 9
(45)
mix Cell suspension
Premix(2)
<4P3-3>


No.1~3 No.1~3<
Cell suspension 1655 ins①~③ ara(+)
1655 ins①~③ ara(-)
Premix① GXL Buffer 30
dNTP 12
GXL 3
(45)
Premix②
Premix(1) 21 21
FimE-C2-EcoRI 7 7
TetP1 7
TetP2 7
(35) (35)
mix Premix② 5
Cell suspension 5
<4P3-3>


LB + arabinose (0.2%)plating (single colony isolation)
1655 + ins①~③ No.1~3
1655 No.1~3
LB + IPTG (0.4g/ml) plating (single colony isolation)
1655 + ins①~③ No.1~3
1655 No.1~3
LB plating (single colony isolation)
1655 + ins①~③ No.1~3
1655 No.1~3


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