From 2010.igem.org
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| | + | <a href="Methods"><img src="https://static.igem.org/mediawiki/2010/6/6f/Method_button.png" width = 300></a> |
| | + | <a href="Methods/PCR"><img src="https://static.igem.org/mediawiki/2010/e/e2/PCR-KAIST3.png" width = 300></a> |
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| | <span style=font-size:30px><font face="Maiandra GD"><b>PCR Strategy</b></font></span> | | <span style=font-size:30px><font face="Maiandra GD"><b>PCR Strategy</b></font></span> |
Revision as of 08:54, 13 October 2010
|
PCR Strategy
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 |
GAS Promoter (expression)
| Primer | Direction | Sequences | Length |
| PP_F (Proximal promoter) | Forward | 5’-CGCCTGCAGGCGAATAGCTGG–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 |
|
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