Team:KAIST-Korea/Notebook/Diary/September

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September 1

pREP41 vector has arrived.

-> we will do E.coli transformation and increase its number.

pREP42-GFP vector has arrived (in E.coli)

-> will incubate a day and do spreading for further experiment.

Making culture media

500mL LB broth + agar 1.5% (7.5g)
Do auto clave
Cool it down until it reaches 50~60℃
Add ampicillin(1000x 100mg/mL) and stir
putting out bubbles on culture media
pouling on petri dish and wait for 1 hour

Culture

Competent cell + DN10A and leave it in ice for 30min
Do heat shock for 45 sec and store it in ice for 2min
Stabilize it in incubator for 30 to 40min
Do spreading

September 2 ~ September 5

PCR Setting

V_STAT1 (expression)

Primer	Direction	Sequences	Length
V_STAT_F	Forward(NdeI)	5’-TGTTCATATGGCTAATGTCTCAGTGGTACGAACTTCAG-3’	24bp
V_STAT_R	Reverse(BamHI)	5’-TATCGGATCCGAATTTACACTTCAGACACAGAAATCAAC-3’	25bp

Materials	Amount
STAT1(KRIBB) (template)	150pmole
V_STAT_F	50pmole
V_STAT_R	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

STAT1 (submission)

Primer	Direction	Sequences	Length
STAT_F	Forward	5’-ATGTCTCAGTGGTACGAACTTCAG-3’	24bp
STAT_R	Reverse	5’-TTACACTTCAGACACAGAAATCAAC-3’	25bp

Materials	Amount
STAT1(KRIBB) (template)	150pmole
STAT_F	50pmole
STAT_R	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

Fusion Antibody Receptor (Submission)

Primer	Direction	Sequences	Length
SIG_F	Forward	5’-ATGGTCTTTTTAAATTCCTCTCCC-3’	24bp
Ab_R	Reverse	5’-GGGGCTGTTGTTTTGGCTGAGG-3’	22bp

Materials	Amount
Fusion antibody receptor T vector (template)	150pmole
SIG_F	50pmole
Ab_R	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

Signal Peptide (Submission)

Primer	Direction	Sequences	Length
SIG_F	Forward	5’-ATGGTCTTTTTAAATTCCTCTCCC-3’	24bp
SIG_R	Reverse	5’-AGCCGCCACCAACCGAGTAGAAA-3’	23bp

Materials	Amount
Fusion antibody receptor T vector (template)	150pmole
SIG_F	50pmole
SIG_R	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

Ig-like (Submission)

Primer	Direction	Sequences	Length
IG_F	Forward	5’-AGGCCGTCCCCGACCTTGCCTG-3’	22bp
IG_R	Reverse	5’-GGAGTCATCATCATCATCATCATC-3’	24bp

Materials	Amount
Fusion antibody receptor T vector (template)	150pmole
IG_F	50pmole
IG_R	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

Signal Peptide + Ig-like (Submission)

Primer	Direction	Sequences	Length
SIG_F	Forward	5’-ATGGTCTTTTTAAATTCCTCTCCC-3’	24bp
IG_R	Reverse	5’-GGAGTCATCATCATCATCATCATC-3’	24bp

Materials	Amount
Fusion antibody receptor T vector (template)	150pmole
SIG_F	50pmole
IG_R	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

Antibody (Submission)

Primer	Direction	Sequences	Length
Ab_F	Forward	5’-ACCCAGTCTCCAGCAATCATGTC-3’	23bp
Ab_R	Reverse	5’-GGGGCTGTTGTTTTGGCTGAGG-3’	22bp

Materials	Amount
Fusion antibody receptor T vector (template)	150pmole
Ab_F	50pmole
Ab_R	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

V_Fusion Antibody Receptor (Expression)

Primer	Direction	Sequences	Length
SIG_F	Forward	5’-ATGGTCTTTTTAAATTCCTCTCCC-3’	24bp
Linker_R	Reverse	5’-CTCTCTTCCAGGGCTTCCAGAACGGGGCTGTTGTTTTGGCTGAGGA-3’	46(23+23)bp

Materials	Amount
Fusion antibody receptor T vector (template)	150pmole
SIG_F	50pmole
Linker_R	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

B_FGFR1 (Submission)

Primer	Direction	Sequences	Length
B_FGFR_F	Forward	5’-GTTCTGGAAGCCCTGGAAGAGAG-3’	23bp
B_FGFR_R	Reverse	5’-TCAGCGGCGTTTGAGTCCGCCAT-3’	23bp

Materials	Amount
FGFR1(KRIBB) (template)	150pmole
B_FGFR_F	50pmole
B_FGFR_R	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

VB_FGFR1 (Expression)

Primer	Direction	Sequences	Length
Linker_F	Forward	5’-TCCTCAGCCAAAACAACAGCCCCGTTCTGGAAGCCCTGGAAGAGAG-3’	46(23+23)bp
B_FGFR_R	Reverse	5’-TCAGCGGCGTTTGAGTCCGCCAT-3’	23bp

Materials	Amount
FGFR1(KRIBB) (template)	150pmole
Linker_F	50pmole
B_FGFR_R	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

Fusion Antibody Receptor (Expression)

Primer	Direction	Sequences	Length
SIG_F	Forward	5’-ATGGTCTTTTTAAATTCCTCTCCC-3’	24bp
B_FGFR_R	Reverse	5’-TCAGCGGCGTTTGAGTCCGCCAT-3’	23bp

Materials	Amount
V_Fusion antibody receptor (template)	150pmole
VB_FGFR1(2+3) (template)	150pmole
SIG_F	50pmole
B_FGFR_R	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

5’UTR (expression)

Primer	Direction	Sequences	Length
HR_F	Forward	5’-AAACCAGTTTTGCCAAAACAAC-3’	22bp
UF_LinkR	Reverse	5’ –GAGAGGAATTTAAAAAGACCATAAACAACATTTTTCTTTTTTAC - 3’	44(22+22)bp

Materials	Amount
S.pombe genomic DNA (template)	150pmole
HR_F	50pmole
UF_LinkR	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

3’UTR (expression)

Primer	Direction	Sequences	Length
HU_LINKF (HA2 + 3’UTR)	Forward	5’-CCATATGACCCAGATTACGCTTGGAAATTAAATCGTGCGTAAG-3’	43(21+22)bp
HR_R	Reverse	5’ – GCGTTCTTTTTTAATTCTGAATTAA – 3’	25bp

Materials	Amount
S.pombe genomic DNA (template)	150pmole
HU_LINKF (HA2 + 3’UTR)	50pmole
HR_R	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

Selection Marker (expression)

Primer	Direction	Sequences	Length
HS_LINKF (HA1 + Selection Marker)	Forward	5’-CCATATGACCCAGATTACGCTAGCAAGCTTAAGCTTAGCTAC-3’	42(21+21)bp
SH_LINKR	Reverse	5’-GTCAGGAACATCGTATGGGTAGACCTGCAGAAGCTTAAGCTTG-3’	43(21+22)bp

Materials	Amount
pREP42-GFP vector (template)	150pmole
HS_LINKF (HA1 + Selection Marker)	50pmole
SH_LINKR	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

Core promoter + GFP (expression)

Primer	Direction	Sequences	Length
PC_LINKF (Proximal + Core)	Forward	5’–CAGAATAGACACACGGGCCGACATTGAAGATATATAAAGGAAG–3’	43(20+23)bp
CGFP_R (Core promoter + GFP)	Reverse	3’-CGACATATGCTTGTACAGCTCG-5’	22bp

Materials	Amount
pREP42-GFP vector (template)	150pmole
PC_LINKF (Proximal + Core)	50pmole
CGFP_R (Core promoter + GFP)	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

Proximal promoter (expression)

Primer	Direction	Sequences	Length
PP_F (Proximal promoter)	Forward	5’-CGCCTGCAGGCGAATAGCTGG–3’	21bp
PC_LINKR	Reverse	5’–CTTCCTTTATATATCTTCAATGTCGGCCCGTGTGTCTATTCTG–3’	43(23+20)bp

Materials	Amount
Proximal promoter T vector (template)	150pmole
PP_F (Proximal promoter)	50pmole
PC_LINKR	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

HA1 (expression)

Primer	Direction	Sequences	Length
FH_LINKF (FGFR + HA1)	Forward	5’-GCGGACTCAAACGCCGCTGATACCCATACGATGTTCCTGAC-3’	41(20+21)bp
HS_LINKR	Reverse	5’-GTAGCTAAGCTTAAGCTTGCTAGCGTAATCTGGGTCATATGG-3’	42(21+21)bp

Materials	Amount
HA T vector (template)	150pmole
FH_LINKF (FGFR + HA1)	50pmole
HS_LINKR	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

HA2 (expression)

Primer	Direction	Sequences	Length
SH_LINKF (Selection Marker + HA2)	Forward	5’-CAAGCTTAAGCTTCTGCAGGTCTACCCATACGATGTTCCTGAC-3’	43(22+21)bp
HU_LINKR	Reverse	5’-CTTACGCACGATTTAATTTCCAAGCGTAATCTGGGTCATATGG-3’	43(22+21)bp

Materials	Amount
HA T vector (template)	150pmole
SH_LINKF (Selection Marker + HA2)	50pmole
HU_LINKR	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

Fusion antibody receptor (expression)

Primer	Direction	Sequences	Length
UF_LinkF (5’UTR+FGFR Linker)	Forward	5’-GTAAAAAAGAAAAATGTTGTTTATGGTCTTTTTAAATTCCTCTC-3’	44(22+22)bp
FH_LINKR	Reverse	5’-GTCAGGAACATCGTATGGGTATCAGCGGCGTTTGAGTCCGC-3’	41(21+20)bp

Materials	Amount
Fusion Antibody receptor (template)	150pmole
UF_LinkF (5’UTR+FGFR Linker)	50pmole
FH_LINKR	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

5’UTR + Fusion Antibody receptor (expression)

Primer	Direction	Sequences	Length
HR_F (for homologous recombinant)	Forward	5’-AAACCAGTTTTGCCAAAACAAC-3’	22bp
FH_LINKR	Reverse	5’-GTCAGGAACATCGTATGGGTATCAGCGGCGTTTGAGTCCGC-3’	41(21+20)bp

Materials	Amount
5'UTR (template)	150pmole
Fusion Antibody receptor (template)	150pmole
FH_LINKF (FGFR + HA1)	50pmole
SH_LINKR	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

HA1 + Selection Marker (expression)

Primer	Direction	Sequences	Length
FH_LINKF (FGFR + HA1)	Forward	5’-GCGGACTCAAACGCCGCTGATACCCATACGATGTTCCTGAC-3’	41(20+21)bp
SH_LINKR	Reverse	5’-GTCAGGAACATCGTATGGGTAGACCTGCAGAAGCTTAAGCTTG-3’	43(21+22)bp

Materials	Amount
HA1 (template)	150pmole
Selection Marker (template)	150pmole
FH_LINKF (FGFR + HA1)	50pmole
SH_LINKR	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

HA2 + 3'UTR (expression)

Primer	Direction	Sequences	Length
SH_LINKF (Selection Marker + HA2)	Forward	5’-CAAGCTTAAGCTTCTGCAGGTCTACCCATACGATGTTCCTGAC-3’	43(22+21)bp
HR_R	Reverse	5’ – GCGTTCTTTTTTAATTCTGAATTAA – 3’	25bp

Materials	Amount
HA2 (template)	150pmole
3’UTR (template)	150pmole
SH_LINKF (Selection Marker + HA2)	50pmole
HR_R	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

5’UTR - Fusion Antibody receptor + HA1 - Selection Marker (expression)

Primer	Direction	Sequences	Length
HR_F(for homologous recombinant)	Forward	5’-AAACCAGTTTTGCCAAAACAAC-3’	22bp
SH_LINKR	Reverse	5’-GTCAGGAACATCGTATGGGTAGACCTGCAGAAGCTTAAGCTTG-3’	43(21+22)bp

Materials	Amount
5’UTR - Fusion Antibody receptor (template)	150pmole
HA1 - Selection Marker (template)	150pmole
HR_F (for homologous recombinant)	50pmole
SH_LINKR	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

Homologous Recombinant (expression)

Primer	Direction	Sequences	Length
HR_F(for homologous recombinant)	Forward	5’-AAACCAGTTTTGCCAAAACAAC-3’	22bp
HR_R	Reverse	5’ – GCGTTCTTTTTTAATTCTGAATTAA – 3’	25bp

Materials	Amount
HA2 + 3'UTR (template)	150pmole
5’UTR - Fusion Antibody receptor - HA1 - Selection Marker (template)	150pmole
HR_F (for homologous recombinant)	50pmole
HR_R	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

September 6 ~ Septembr 9

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	-	-

September 10 ~ September 12

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	-	-

September 13 ~ September 16

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

</tr>

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