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Team:KAIST-Korea/Project/Methods/PCR
From 2010.igem.org
Revision as of 08:56, 13 October 2010 by
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PCR Strategy
V_STAT1 (expression)
Primer
Direction
Sequences
Length
V_STAT_F
Forward(NdeI)
5’-
TGTT
CATATG
GCTA
ATGTCTCAGTGGTACGAACTTCAG-3’
24bp
V_STAT_R
Reverse(BamHI)
5’-
TATC
GGATCC
GAAT
TTACACTTCAGACACAGAAATCAAC-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’-
CGA
CATATG
CTT
GTACAGCTCG-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’-
CGC
CTGCAG
GCG
AATAGCTGG–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
GAS Promoter (expression)
Primer
Direction
Sequences
Length
PP_F (Proximal promoter)
Forward
5’-
CGC
CTGCAG
GCG
AATAGCTGG–3’
21bp
CGFP_R (Core promoter + GFP)
Reverse
3’-CGACATATGCTTGTACAGCTCG-5’
22bp
Materials
Amount
Proximal promoter(template)
150pmole
Core promoter + GFP(template)
150pmole
PP_F (Proximal promoter)
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
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