Team:KAIST-Korea/Project/Methods/PCR Result

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PCR Experiment

9/6 ~ 9/9 PCR Result

PCR composition

Materials	Amount
Template	100pmole
Primer A	50pmole
Primer B	50pmole
dNTP (dNTP mixture 2.5mM TaKaRa)	2ul
Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE)	0.25ul
10x buffer (10x cloned Pfu Reaction buffer)	5ul
DW (3rd sterile water)	rest
Total	50ul

Exp 1

Segment

Annealing Temperature

Result

a	STAT1	53*C	-	-
b	V_STAT1	53*C	-	-
c	Fusion Antibody receptor	53*C	-	-
d	Signal peptide	56*C	-	-
e	Ig-like	56*C	-	-
f	Signal Peptide + Ig-like	56*C	-	-
g	Antibody	56*C	-	-
h	V_Fusion Antibody Receptor	53*C	-	-
i	B_FGFR1	56*C	-	-
j	VB_FGFR1	56*C	-	-

Exp 2

Segment

Annealing Temperature

Result

a	STAT1	48*C	60%	2139bp
b	V_STAT1	48*C	-	-

Exp 3

Segment

Annealing Temperature

Result

a	STAT1	50*C	100%	2139bp
b	V_STAT1	50*C	-	-

Exp 4

Segment

Annealing Temperature

Result

a	Fusion Antibody receptor	50*C	-	-
b	Signal peptide	50*C	-	-
c	Ig-like	50*C	60%	339bp
d	Signal Peptide + Ig-like	50*C	-	-
e	Antibody	50*C	-	-
f	V_Fusion Antibody Receptor	50*C	-	-

9/10 ~ 9/12 PCR Result

PCR composition

Materials	Amount
Template	150pmole
Primer A	50pmole
Primer B	50pmole
dNTP (dNTP mixture 2.5mM TaKaRa)	5ul
Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE)	0.25ul
10x buffer (10x cloned Pfu Reaction buffer)	5ul
DW (3rd sterile water)	rest
Total	50ul

Exp 1

Segment

Annealing Temperature

Result

a	STAT1	60*C	-	-
b	V_STAT1	60*C	-	-
c	Fusion Antibody receptor	60*C	70%	1095bp
d	Signal peptide	60*C	-	-
e	Ig-like	60*C	60%	339bp
f	Signal Peptide + Ig-like	60*C	-	-
g	Antibody	60*C	100%	669bp
h	V_Fusion Antibody Receptor	60*C	-	-
i	B_FGFR1	60*C	-	-
j	VB_FGFR1	60*C	-	-
x1	negative 1	60*C	-	-
x2	negative 2	60*C	-	-

Exp 2

Segment

Annealing Temperature

Result

a	STAT1	58*C	60%	2139bp
b	V_STAT1	58*C	-	-
c	Fusion Antibody receptor	58*C	80%	1095bp
d	Signal peptide	58*C	80%	87bp
e	Ig-like	58*C	100%	339bp
f	Signal Peptide + Ig-like	58*C	80%	426bp
g	Antibody	58*C	100%	669bp
h	V_Fusion Antibody Receptor	58*C	-	-
i	B_FGFR1	58*C	-	-
j	VB_FGFR1	58*C	-	-
x	negative	58*C	-	-

Exp 3

Segment

Annealing Temperature

Result

a	STAT1	62*C	-	-
b	V_STAT1	62*C	-	-
c	Fusion Antibody receptor	62*C	10%	1095bp
d	Signal peptide	62*C	-	-
e	Ig-like	62*C	-	-
f	Signal Peptide + Ig-like	62*C	-	-
g	Antibody	62*C	-	-
h	V_Fusion Antibody Receptor	62*C	-	-
i	B_FGFR1	62*C	-	-
j	VB_FGFR1	62*C	-	-
x	negative	62*C	-	-

Exp 4

Segment

Annealing Temperature

Result

a	STAT1	56*C	-	-
b	V_STAT1	56*C	-	-
c	Fusion Antibody receptor	56*C	5%	1095bp
d	Signal peptide	56*C	-	-
e	Ig-like	56*C	80%	339bp
f	Signal Peptide + Ig-like	56*C	-	-
g	Antibody	56*C	80%	669bp
h	V_Fusion Antibody Receptor	56*C	-	-
i	B_FGFR1	56*C	-	-
j	VB_FGFR1	56*C	-	-
x	negative	56*C	-	-

Exp 5

Segment

Annealing Temperature

Result

a	V_STAT1	64*C	-	-
b	B_FGFR1	64*C	5%	1398bp
c	VB_FGFR1	64*C	-	-

Exp 6

Segment

Annealing Temperature

Result

a	V_STAT1	64*C	-	-
b	B_FGFR1	64*C	3%	1398bp
c	VB_FGFR1	64*C	-	-

Exp 7

Segment

Annealing Temperature

Result

a	3'UTR	62*C	-	-
b	5'UTR	62*C	-	-

Exp 8

Segment

Annealing Temperature

Result

a	V_STAT1	68*C	-	-
b	B_FGFR1	68*C	10%	1398bp
c	VB_FGFR1	68*C	-	-

Exp 9

Segment

Annealing Temperature

Result

a	V_STAT1	70*C	-	-
b	B_FGFR1	70*C	3%	1398bp
c	VB_FGFR1	70*C	-	-
c	5'UTR	70*C	-	-
c	3'UTR	70*C	-	-

Exp 10

Segment

Annealing Temperature

Result

a	V_STAT1	67*C	-	-
b	B_FGFR1	67*C	6%	1398bp
c	VB_FGFR1	67*C	-	-

9/13 ~ 9/16 PCR Result

PCR composition

Materials	Amount
Template	100pmole
Primer A	50pmole
Primer B	50pmole
Bioneer Accupower HF PCR premix	< 1ul
DW (3rd sterile water)	rest
Total	50ul

Exp 1

Segment

Annealing Temperature

Result

a	STAT1	50*C	100%	2139bp
b	STAT1	52*C	100%	2139bp
c	STAT1	54*C	100%	2139bp
d	STAT1	56*C	100%	2139bp
e	STAT1	58*C	100%	2139bp
f	STAT1	60*C	100%	2139bp
g	V_STAT1	50*C	100%	2151bp
h	V_STAT1	52*C	100%	2151bp
i	V_STAT1	54*C	100%	2151bp
j	V_STAT1	56*C	100%	2151bp
k	V_STAT1	58*C	100%	2151bp
l	V_STAT1	60*C	100%	2151bp

Exp 2

Segment

Annealing Temperature

Result

a	B_FGFR	50*C	100%	1398bp
b	B_FGFR	52*C	100%	1398bp
c	B_FGFR	54*C	100%	1398bp
d	B_FGFR	56*C	100%	1398bp
e	B_FGFR	58*C	100%	1398bp
f	B_FGFR	60*C	100%	1398bp
g	VB_FGFR	50*C	100%	1420bp
h	VB_FGFR	52*C	100%	1420bp
i	VB_FGFR	54*C	100%	1420bp
j	VB_FGFR	56*C	100%	1420bp
k	VB_FGFR	58*C	100%	1420bp
l	VB_FGFR	60*C	100%	1420bp

Exp 3

Segment

Annealing Temperature

Result

a	Signal peptide	61*C	30%	87bp
b	Ig-like	61*C	100%	339bp
c	Signal peptide + Ig-like	61*C	100%	426bp
d	Signal peptide	59*C	30%	87bp
e	Ig-like	59*C	100%	339bp
f	Signal peptide + Ig-like	59*C	100%	426bp
g	3'UTR	52*C	50%	444bp
h	3'UTR	54*C	50%	444bp
i	3'UTR	56*C	100%	444bp
j	3'UTR	58*C	100%	444bp
k	3'UTR	60*C	100%	444bp
l	5'UTR	52*C	100%	500bp
m	5'UTR	54*C	100%	500bp
n	5'UTR	56*C	100%	500bp
o	5'UTR	58*C	100%	500bp
p	5'UTR	69*C	70%	500bp

Exp 4

Segment

Annealing Temperature

Result

a	F_Fusion antibody receptor	60*C	100%	1095bp
b	F_Fusion antibody receptor	58*C	100%	1095bp
c	Antibody	60*C	100%	669bp
d	Antibody	58*C	100%	669bp
e	VF_Fusion antibody receptor	60*C	100%	1107bp
f	VF_Fusion antibody receptor	58*C	100%	1107bp

9/17 PCR result

PCR composition

Materials	Amount
Template	100pmole
Primer A	50pmole
Primer B	50pmole
Bioneer Accupower HF PCR premix	< 1ul
DW (3rd sterile water)	rest
Total	50ul

Exp 1

Segment

Annealing Temperature

Result

a	Selection marker(41)	50*C	0%	--
b	Selection marker(41)	52*C	0%	--
c	Selection marker(41)	54*C	0%	--
d	Selection marker(41)	56*C	0%	--
e	Selection marker(42)	50*C	0%	--
f	Selection marker(42)	52*C	0%	--
g	Selection marker(42)	54*C	0%	--
h	Selection marker(42)	56*C	0%	--
i	F_Fusion antibody receptor	50*C	100%	1107bp
j	F_Fusion antibody receptor	52*C	30%	1107bp
k	B_Fusion antibody receptor	54*C	50%	1398bp
l	B_Fusion antibody receptor	56*C	100%	1398bp

Exp 2

Segment

Annealing Temperature

Result

a	HA1	50*C	100%	87bp
b	HA1	52*C	100%	87bp
c	HA1	54*C	100%	87bp
d	HA1	56*C	100%	87bp
e	HA2	50*C	80%	87bp
f	HA2	52*C	80%	87bp
g	HA2	54*C	80%	87bp
h	HA2	56*C	80%	87bp
i	Proximal promoter	50*C	100%	120bp
j	Proximal promoter	52*C	100%	120bp
k	Proximal promoter	54*C	100%	120bp
l	Proximal promoter	56*C	100%	120bp
m	5' UTR	54*C	100%	500bp
n	5' UTR	54*C	100%	500bp
o	3' UTR	54*C	100%	444bp
p	3' UTR	54*C	100%	444bp

9/18 PCR result

PCR composition

Materials	Amount
Template	100pmole
Primer A	50pmole
Primer B	50pmole
Bioneer Accupower HF PCR premix	< 1ul
DW (3rd sterile water)	rest
Total	50ul

Exp 1

Segment

Annealing Temperature

Result

a	Selection marker(41)	50*C	0%	--
b	Selection marker(41)	52*C	0%	--
c	Selection marker(41)	54*C	0%	--
d	Selection marker(41)	56*C	0%	--
e	Selection marker(42)	50*C	0%	--
f	Selection marker(42)	52*C	0%	--
g	Selection marker(42)	54*C	0%	--
h	Selection marker(42)	56*C	0%	--
i	F_Fusion antibody receptor	50*C	100%	1107bp
j	F_Fusion antibody receptor	52*C	30%	1107bp
k	B_Fusion antibody receptor	54*C	50%	1398bp
l	B_Fusion antibody receptor	56*C	100%	1398bp

Exp 2

Segment

Annealing Temperature

Result

a	HA1	50*C	100%	87bp
b	HA1	52*C	100%	87bp
c	HA1	54*C	100%	87bp
d	HA1	56*C	100%	87bp
e	HA2	50*C	80%	87bp
f	HA2	52*C	80%	87bp
g	HA2	54*C	80%	87bp
h	HA2	56*C	80%	87bp
i	Proximal promoter	50*C	100%	120bp
j	Proximal promoter	52*C	100%	120bp
k	Proximal promoter	54*C	100%	120bp
l	Proximal promoter	56*C	100%	120bp
m	5' UTR	54*C	100%	500bp
n	5' UTR	54*C	100%	500bp
o	3' UTR	54*C	100%	444bp
p	3' UTR	54*C	100%	444bp

9/19 PCR result

PCR composition

Materials	Amount
Template	100pmole
Primer A	50pmole
Primer B	50pmole
Bioneer Accupower HF PCR premix	< 1ul
DW (3rd sterile water)	rest
Total	50ul

Exp 1

Segment

Annealing Temperature

Result

a	Selection marker(41)	50*C	0%	--
b	Selection marker(41)	52*C	0%	--
c	Selection marker(41)	54*C	0%	--
d	Selection marker(41)	56*C	0%	--
e	Selection marker(42)	50*C	0%	--
f	Selection marker(42)	52*C	0%	--
g	Selection marker(42)	54*C	0%	--
h	Selection marker(42)	56*C	0%	--
i	F_Fusion antibody receptor	50*C	100%	1107bp
j	F_Fusion antibody receptor	52*C	30%	1107bp
k	B_Fusion antibody receptor	54*C	50%	1398bp
l	B_Fusion antibody receptor	56*C	100%	1398bp

Exp 2

Segment

Annealing Temperature

Result

a	HA1	50*C	100%	87bp
b	HA1	52*C	100%	87bp
c	HA1	54*C	100%	87bp
d	HA1	56*C	100%	87bp
e	HA2	50*C	80%	87bp
f	HA2	52*C	80%	87bp
g	HA2	54*C	80%	87bp
h	HA2	56*C	80%	87bp
i	Proximal promoter	50*C	100%	120bp
j	Proximal promoter	52*C	100%	120bp
k	Proximal promoter	54*C	100%	120bp
l	Proximal promoter	56*C	100%	120bp
m	5' UTR	54*C	100%	500bp
n	5' UTR	54*C	100%	500bp
o	3' UTR	54*C	100%	444bp
p	3' UTR	54*C	100%	444bp

9/21 PCR result

PCR composition

Materials	Amount
Template	100pmole
Primer A	50pmole
Primer B	50pmole
Bioneer Accupower HF PCR premix	< 1ul
DW (3rd sterile water)	rest
Total	50ul

Exp 1

Segment

Annealing Temperature

Result

4

a	Selection marker(41)	50*C	0%	--
b	Selection marker(41)	52*C	0%	--
c	Selection marker(41)	54*C	0%	--
d	Selection marker(41)	56*C	0%	--
e	Selection marker(42)	50*C	0%	--
f	Selection marker(42)	52*C	0%	--
g	Selection marker(42)	54*C	0%	--
h	Selection marker(42)	56*C	0%	--
i	F_Fusion antibody receptor	54*C	30%	1107bp
j	F_Fusion antibody receptor	54*C	50%	1107bp
k	B_Fusion antibody receptor	54*C	100%	1398bp
l	B_Fusion antibody receptor	54*C	100%	1398bp

Exp 2

Segment

Annealing Temperature

Result

a	HA1	50*C	100%	87bp
b	HA1	52*C	100%	87bp
c	HA1	54*C	100%	87bp
d	HA1	56*C	100%	87bp
e	HA2	50*C	80%	87bp
f	HA2	52*C	80%	87bp
g	HA2	54*C	80%	87bp
h	HA2	56*C	80%	87bp
i	Proximal promoter	50*C	100%	120bp
j	Proximal promoter	52*C	100%	120bp
k	Proximal promoter	54*C	100%	120bp
l	Proximal promoter	56*C	100%	120bp
m	5' UTR	54*C	100%	500bp
n	5' UTR	54*C	100%	500bp
o	3' UTR	54*C	100%	444bp
p	3' UTR	54*C	100%	444bp

9/27 PCR result

PCR composition

Materials	Amount
Template	100pmole
Primer A	50pmole
Primer B	50pmole
Bioneer Accupower HF PCR premix	< 1ul
DW (3rd sterile water)	rest
Total	50ul

Exp 1

Segment

Annealing Temperature

Result

4

a	Selection marker(41)	50*C	0%	--
b	Selection marker(41)	52*C	0%	--
c	Selection marker(41)	54*C	0%	--
d	Selection marker(41)	56*C	0%	--
e	Selection marker(42)	50*C	0%	--
f	Selection marker(42)	52*C	0%	--
g	Selection marker(42)	54*C	0%	--
h	Selection marker(42)	56*C	0%	--
i	F_Fusion antibody receptor	54*C	30%	1107bp
j	F_Fusion antibody receptor	54*C	50%	1107bp
k	B_Fusion antibody receptor	54*C	100%	1398bp
l	B_Fusion antibody receptor	54*C	100%	1398bp

Exp 2

Segment

Annealing Temperature

Result

a	HA1	50*C	100%	87bp
b	HA1	52*C	100%	87bp
c	HA1	54*C	100%	87bp
d	HA1	56*C	100%	87bp
e	HA2	50*C	80%	87bp
f	HA2	52*C	80%	87bp
g	HA2	54*C	80%	87bp
h	HA2	56*C	80%	87bp
i	Proximal promoter	50*C	100%	120bp
j	Proximal promoter	52*C	100%	120bp
k	Proximal promoter	54*C	100%	120bp
l	Proximal promoter	56*C	100%	120bp
m	5' UTR	54*C	100%	500bp
n	5' UTR	54*C	100%	500bp
o	3' UTR	54*C	100%	444bp
p	3' UTR	54*C	100%	444bp

9/28 PCR result

PCR composition

Materials	Amount
Template	100pmole
Primer A	50pmole
Primer B	50pmole
Bioneer Accupower HF PCR premix	< 1ul
DW (3rd sterile water)	rest
Total	50ul

Exp 1

Segment

Annealing Temperature

Result

4

a	Selection marker(41)	50*C	0%	--
b	Selection marker(41)	52*C	0%	--
c	Selection marker(41)	54*C	0%	--
d	Selection marker(41)	56*C	0%	--
e	Selection marker(42)	50*C	0%	--
f	Selection marker(42)	52*C	0%	--
g	Selection marker(42)	54*C	0%	--
h	Selection marker(42)	56*C	0%	--
i	F_Fusion antibody receptor	54*C	30%	1107bp
j	F_Fusion antibody receptor	54*C	50%	1107bp
k	B_Fusion antibody receptor	54*C	100%	1398bp
l	B_Fusion antibody receptor	54*C	100%	1398bp

Exp 2

Segment

Annealing Temperature

Result

a	HA1	50*C	100%	87bp
b	HA1	52*C	100%	87bp
c	HA1	54*C	100%	87bp
d	HA1	56*C	100%	87bp
e	HA2	50*C	80%	87bp
f	HA2	52*C	80%	87bp
g	HA2	54*C	80%	87bp
h	HA2	56*C	80%	87bp
i	Proximal promoter	50*C	100%	120bp
j	Proximal promoter	52*C	100%	120bp
k	Proximal promoter	54*C	100%	120bp
l	Proximal promoter	56*C	100%	120bp
m	5' UTR	54*C	100%	500bp
n	5' UTR	54*C	100%	500bp
o	3' UTR	54*C	100%	444bp
p	3' UTR	54*C	100%	444bp

10/1 PCR result

PCR composition

Materials	Amount
Template	100pmole
Primer A	50pmole
Primer B	50pmole
Bioneer Accupower HF PCR premix	< 1ul
DW (3rd sterile water)	rest
Total	50ul

Exp 1

Segment

Annealing Temperature

Result

4

a	Selection marker(41)	50*C	0%	--
b	Selection marker(41)	52*C	0%	--
c	Selection marker(41)	54*C	0%	--
d	Selection marker(41)	56*C	0%	--
e	Selection marker(42)	50*C	0%	--
f	Selection marker(42)	52*C	0%	--
g	Selection marker(42)	54*C	0%	--
h	Selection marker(42)	56*C	0%	--
i	F_Fusion antibody receptor	54*C	30%	1107bp
j	F_Fusion antibody receptor	54*C	50%	1107bp
k	B_Fusion antibody receptor	54*C	100%	1398bp
l	B_Fusion antibody receptor	54*C	100%	1398bp

Exp 2

Segment

Annealing Temperature

Result

a	HA1	50*C	100%	87bp
b	HA1	52*C	100%	87bp
c	HA1	54*C	100%	87bp
d	HA1	56*C	100%	87bp
e	HA2	50*C	80%	87bp
f	HA2	52*C	80%	87bp
g	HA2	54*C	80%	87bp
h	HA2	56*C	80%	87bp
i	Proximal promoter	50*C	100%	120bp
j	Proximal promoter	52*C	100%	120bp
k	Proximal promoter	54*C	100%	120bp
l	Proximal promoter	56*C	100%	120bp
m	5' UTR	54*C	100%	500bp
n	5' UTR	54*C	100%	500bp
o	3' UTR	54*C	100%	444bp
p	3' UTR	54*C	100%	444bp

10/5 PCR result

PCR composition

Materials	Amount
Template	100pmole
Primer A	50pmole
Primer B	50pmole
Bioneer Accupower HF PCR premix	< 1ul
DW (3rd sterile water)	rest
Total	50ul

Exp 1

Segment

Annealing Temperature

Result

4

a	Selection marker(41)	50*C	0%	--
b	Selection marker(41)	52*C	0%	--
c	Selection marker(41)	54*C	0%	--
d	Selection marker(41)	56*C	0%	--
e	Selection marker(42)	50*C	0%	--
f	Selection marker(42)	52*C	0%	--
g	Selection marker(42)	54*C	0%	--
h	Selection marker(42)	56*C	0%	--
i	F_Fusion antibody receptor	54*C	30%	1107bp
j	F_Fusion antibody receptor	54*C	50%	1107bp
k	B_Fusion antibody receptor	54*C	100%	1398bp
l	B_Fusion antibody receptor	54*C	100%	1398bp

Exp 2

Segment

Annealing Temperature

Result

a	HA1	50*C	100%	87bp
b	HA1	52*C	100%	87bp
c	HA1	54*C	100%	87bp
d	HA1	56*C	100%	87bp
e	HA2	50*C	80%	87bp
f	HA2	52*C	80%	87bp
g	HA2	54*C	80%	87bp
h	HA2	56*C	80%	87bp
i	Proximal promoter	50*C	100%	120bp
j	Proximal promoter	52*C	100%	120bp
k	Proximal promoter	54*C	100%	120bp
l	Proximal promoter	56*C	100%	120bp
m	5' UTR	54*C	100%	500bp
n	5' UTR	54*C	100%	500bp
o	3' UTR	54*C	100%	444bp
p	3' UTR	54*C	100%	444bp