From 2010.igem.org
(Difference between revisions)
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| | === 5’UTR (expression) === | | === 5’UTR (expression) === |
| | === 3’UTR (expression) === | | === 3’UTR (expression) === |
| | + | |
| | + | <center> |
| | + | [[Image:PCR_HR.jpg]] |
| | + | |
| | + | <br> |
| | + | |
| | + | <table border=1> |
| | + | <tr align=center> |
| | + | <td><b>Primer</b></td><td><b>Direction</b></td><td><b>Sequences</b></td><td><b>Length</b></td> |
| | + | </tr> |
| | + | <tr align=center> |
| | + | <td>HU_LINKF (HA2 + 3’UTR)</td><td>Forward</td><td>5’-CCATATGACCCAGATTACGCTTGGAAATTAAATCGTGCGTAAG-3’</td><td>43bp</td> |
| | + | </tr> |
| | + | <tr align=center> |
| | + | <td>HR_R</td><td>Reverse</td><td>5’ – GCGTTCTTTTTTAATTCTGAATTAA – 3’</td><td>25bp</td> |
| | + | </tr> |
| | + | </table> |
| | + | |
| | + | <br> |
| | + | |
| | + | <table border=1> |
| | + | <tr align=center> |
| | + | <td><b>Materials</b></td><td><b>Volume</b></td> |
| | + | </tr> |
| | + | <tr align=center> |
| | + | <td>S.pombe genomic DNA (template)</td><td></td> |
| | + | </tr> |
| | + | <tr align=center> |
| | + | <td>HU_LINKF (HA2 + 3’UTR)</td><td></td> |
| | + | </tr> |
| | + | <tr align=center> |
| | + | <td>HR_R</td><td></td> |
| | + | </tr> |
| | + | <tr align=center> |
| | + | <td>dNTP (dNTP mixture 2.5mM TaKaRa)</td><td></td> |
| | + | </tr> |
| | + | <tr align=center> |
| | + | <td>Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE)</td><td></td> |
| | + | </tr> |
| | + | <tr align=center> |
| | + | <td>10x buffer (10x cloned Pfu Reaction buffer)</td><td></td> |
| | + | </tr> |
| | + | <tr align=center> |
| | + | <td>DW (3rd sterile water)</td><td></td> |
| | + | </tr> |
| | + | <tr align=center> |
| | + | <td>Total</td><td>50ul</td> |
| | + | </tr> |
| | + | </table> |
| | + | |
| | + | [[Image:PCR_cycle.jpg]] |
| | + | </center> |
| | + | <br> |
| | + | <br> |
| | + | <br> |
| | + | |
| | === Selection Marker (expression) === | | === Selection Marker (expression) === |
| | | | |
Revision as of 15:07, 10 September 2010
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 ?
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 | Volume |
| STAT1(KRIBB) (template) | |
| V_STAT_F | |
| V_STAT_R | |
| dNTP (dNTP mixture 2.5mM TaKaRa) | |
| Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE) | |
| 10x buffer (10x cloned Pfu Reaction buffer) | |
| DW (3rd sterile water) | |
| Total | 50ul |
STAT1 (submission)
| Primer | Direction | Sequences | Length |
| STAT_F | Forward | 5’-ATGTCTCAGTGGTACGAACTTCAG-3’ | 24bp |
| STAT_R | Reverse | 5’-TTACACTTCAGACACAGAAATCAAC-3’ | 25bp |
| Materials | Volume |
| STAT1(KRIBB) (template) | |
| STAT_F | |
| STAT_R | |
| dNTP (dNTP mixture 2.5mM TaKaRa) | |
| Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE) | |
| 10x buffer (10x cloned Pfu Reaction buffer) | |
| DW (3rd sterile water) | |
| 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 | Volume |
| Fusion antibody receptor T vector (template) | |
| SIG_F | |
| Ab_R | |
| dNTP (dNTP mixture 2.5mM TaKaRa) | |
| Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE) | |
| 10x buffer (10x cloned Pfu Reaction buffer) | |
| DW (3rd sterile water) | |
| Total | 50ul |
Signal Peptide (Submission)
| Primer | Direction | Sequences | Length |
| SIG_F | Forward | 5’-ATGGTCTTTTTAAATTCCTCTCCC-3’ | 24bp |
| SIG_R | Reverse | 5’-AGCCGCCACCAACCGAGTAGAAA-3’ | 23bp |
| Materials | Volume |
| Fusion antibody receptor T vector (template) | |
| SIG_F | |
| SIG_R | |
| dNTP (dNTP mixture 2.5mM TaKaRa) | |
| Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE) | |
| 10x buffer (10x cloned Pfu Reaction buffer) | |
| DW (3rd sterile water) | |
| Total | 50ul |
Ig-like (Submission)
| Primer | Direction | Sequences | Length |
| IG_F | Forward | 5’-AGGCCGTCCCCGACCTTGCCTG-3’ | 22bp |
| IG_R | Reverse | 5’-GGAGTCATCATCATCATCATCATC-3’ | 24bp |
| Materials | Volume |
| Fusion antibody receptor T vector (template) | |
| IG_F | |
| IG_R | |
| dNTP (dNTP mixture 2.5mM TaKaRa) | |
| Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE) | |
| 10x buffer (10x cloned Pfu Reaction buffer) | |
| DW (3rd sterile water) | |
| 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 | Volume |
| Fusion antibody receptor T vector (template) | |
| SIG_F | |
| IG_R | |
| dNTP (dNTP mixture 2.5mM TaKaRa) | |
| Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE) | |
| 10x buffer (10x cloned Pfu Reaction buffer) | |
| DW (3rd sterile water) | |
| Total | 50ul |
Antibody (Submission)
| Primer | Direction | Sequences | Length |
| Ab_F | Forward | 5’-ACCCAGTCTCCAGCAATCATGTC-3’ | 23bp |
| Ab_R | Reverse | 5’-GGGGCTGTTGTTTTGGCTGAGG-3’ | 22bp |
| Materials | Volume |
| Fusion antibody receptor T vector (template) | |
| Ab_F | |
| Ab_R | |
| dNTP (dNTP mixture 2.5mM TaKaRa) | |
| Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE) | |
| 10x buffer (10x cloned Pfu Reaction buffer) | |
| DW (3rd sterile water) | |
| 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 | Volume |
| Fusion antibody receptor T vector (template) | |
| SIG_F | |
| Linker_R | |
| dNTP (dNTP mixture 2.5mM TaKaRa) | |
| Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE) | |
| 10x buffer (10x cloned Pfu Reaction buffer) | |
| DW (3rd sterile water) | |
| 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 | Volume |
| FGFR1(KRIBB) (template) | |
| B_FGFR_F | |
| B_FGFR_R | |
| dNTP (dNTP mixture 2.5mM TaKaRa) | |
| Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE) | |
| 10x buffer (10x cloned Pfu Reaction buffer) | |
| DW (3rd sterile water) | |
| 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 | Volume |
| FGFR1(KRIBB) (template) | |
| Linker_F | |
| B_FGFR_R | |
| dNTP (dNTP mixture 2.5mM TaKaRa) | |
| Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE) | |
| 10x buffer (10x cloned Pfu Reaction buffer) | |
| DW (3rd sterile water) | |
| Total | 50ul |
Fusion Antibody Receptor (Expression)
5’UTR (expression)
3’UTR (expression)
| Primer | Direction | Sequences | Length |
| HU_LINKF (HA2 + 3’UTR) | Forward | 5’-CCATATGACCCAGATTACGCTTGGAAATTAAATCGTGCGTAAG-3’ | 43bp |
| HR_R | Reverse | 5’ – GCGTTCTTTTTTAATTCTGAATTAA – 3’ | 25bp |
| Materials | Volume |
| S.pombe genomic DNA (template) | |
| HU_LINKF (HA2 + 3’UTR) | |
| HR_R | |
| dNTP (dNTP mixture 2.5mM TaKaRa) | |
| Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE) | |
| 10x buffer (10x cloned Pfu Reaction buffer) | |
| DW (3rd sterile water) | |
| Total | 50ul |
Selection Marker (expression)
| Primer | Direction | Sequences | Length |
| HS_LINKF (HA1 + Selection Marker) | Forward | 5’-CCATATGACCCAGATTACGCTAGCAAGCTTAAGCTTAGCTAC-3’ | 42bp |
| SH_LINKR | Reverse | 5’-GTCAGGAACATCGTATGGGTAGACCTGCAGAAGCTTAAGCTTG-3’ | 43bp |
| Materials | Volume |
| pREP42-GFP vector (template) | |
| HS_LINKF (HA1 + Selection Marker) | |
| SH_LINKR | |
| dNTP (dNTP mixture 2.5mM TaKaRa) | |
| Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE) | |
| 10x buffer (10x cloned Pfu Reaction buffer) | |
| DW (3rd sterile water) | |
| Total | 50ul |
Core promoter + GFP (expression)
| Primer | Direction | Sequences | Length |
| PC_LINKF (Proximal + Core) | Forward | 5’–CAGAATAGACACACGGGCCGACATTGAAGATATATAAAGGAAG–3’ | 43bp |
| CGFP_R (Core promoter + GFP) | Reverse | 3’-CGACATATGCTTGTACAGCTCG-5’ | 22bp |
| Materials | Volume |
| pREP42-GFP vector (template) | |
| PC_LINKF (Proximal + Core) | |
| CGFP_R (Core promoter + GFP) | |
| dNTP (dNTP mixture 2.5mM TaKaRa) | |
| Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE) | |
| 10x buffer (10x cloned Pfu Reaction buffer) | |
| DW (3rd sterile water) | |
| Total | 50ul |
Proximal promoter (expression)
| Primer | Direction | Sequences | Length |
| PP_F (Proximal promoter) | Forward | 5’-CGCCTGCAGGCGAATAGCTGG–3’ | 41bp |
| PC_LINKR | Reverse | 5’–CTTCCTTTATATATCTTCAATGTCGGCCCGTGTGTCTATTCTG–3’ | 43bp |
| Materials | Volume |
| Proximal promoter T vector (template) | |
| PP_F (Proximal promoter) | |
| PC_LINKR | |
| dNTP (dNTP mixture 2.5mM TaKaRa) | |
| Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE) | |
| 10x buffer (10x cloned Pfu Reaction buffer) | |
| DW (3rd sterile water) | |
| Total | 50ul |
HA1 (expression)
| Primer | Direction | Sequences | Length |
| FH_LINKF (FGFR + HA1) | Forward | 5’-GCGGACTCAAACGCCGCTGATACCCATACGATGTTCCTGAC-3’ | 41bp |
| HS_LINKR | Reverse | 5’-GTAGCTAAGCTTAAGCTTGCTAGCGTAATCTGGGTCATATGG-3’ | 42bp |
| Materials | Volume |
| HA T vector (template) | |
| FH_LINKF (FGFR + HA1) | |
| HS_LINKR | |
| dNTP (dNTP mixture 2.5mM TaKaRa) | |
| Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE) | |
| 10x buffer (10x cloned Pfu Reaction buffer) | |
| DW (3rd sterile water) | |
| Total | 50ul |
HA2 (expression)
| Primer | Direction | Sequences | Length |
| SH_LINKF (Selection Marker + HA2) | Forward | 5’-CAAGCTTAAGCTTCTGCAGGTCTACCCATACGATGTTCCTGAC-3’ | 43bp |
| HU_LINKR | Reverse | 5’-CTTACGCACGATTTAATTTCCAAGCGTAATCTGGGTCATATGG-3’ | 43bp |
| Materials | Volume |
| HA T vector (template) | |
| SH_LINKF (Selection Marker + HA2) | |
| HU_LINKR | |
| dNTP (dNTP mixture 2.5mM TaKaRa) | |
| Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE) | |
| 10x buffer (10x cloned Pfu Reaction buffer) | |
| DW (3rd sterile water) | |
| Total | 50ul |
Fusion antibody receptor (expression)
| Primer | Direction | Sequences | Length |
| UF_LinkF (5’UTR+FGFR Linker) | Forward | 5’-GTAAAAAAGAAAAATGTTGTTTATGGTCTTTTTAAATTCCTCTC-3’ | 44bp |
| FH_LINKR | Reverse | 5’-GTCAGGAACATCGTATGGGTATCAGCGGCGTTTGAGTCCGC-3’ | 41bp |
| Materials | Volume |
| Fusion Antibody receptor (template) | |
| UF_LinkF (5’UTR+FGFR Linker) | |
| FH_LINKR | |
| dNTP (dNTP mixture 2.5mM TaKaRa) | |
| Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE) | |
| 10x buffer (10x cloned Pfu Reaction buffer) | |
| DW (3rd sterile water) | |
| 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’ | 41bp |
| Materials | Volume |
| 5'UTR (template) | |
| Fusion Antibody receptor (template) | |
| FH_LINKF (FGFR + HA1) | |
| SH_LINKR | |
| dNTP (dNTP mixture 2.5mM TaKaRa) | |
| Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE) | |
| 10x buffer (10x cloned Pfu Reaction buffer) | |
| DW (3rd sterile water) | |
| Total | 50ul |
HA1 + Selection Marker (expression)
| Primer | Direction | Sequences | Length |
| FH_LINKF (FGFR + HA1) | Forward | 5’-GCGGACTCAAACGCCGCTGATACCCATACGATGTTCCTGAC-3’ | 41bp |
| SH_LINKR | Reverse | 5’-GTCAGGAACATCGTATGGGTAGACCTGCAGAAGCTTAAGCTTG-3’ | 43bp |
| Materials | Volume |
| HA1 (template) | |
| Selection Marker (template) | |
| FH_LINKF (FGFR + HA1) | |
| SH_LINKR | |
| dNTP (dNTP mixture 2.5mM TaKaRa) | |
| Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE) | |
| 10x buffer (10x cloned Pfu Reaction buffer) | |
| DW (3rd sterile water) | |
| Total | 50ul |
HA2 + 3'UTR (expression)
| Primer | Direction | Sequences | Length |
| SH_LINKF (Selection Marker + HA2) | Forward | 5’-CAAGCTTAAGCTTCTGCAGGTCTACCCATACGATGTTCCTGAC-3’ | 43bp |
| HR_R | Reverse | 5’ – GCGTTCTTTTTTAATTCTGAATTAA – 3’ | 25bp |
| Materials | Volume |
| HA2 (template) | |
| 3’UTR (template) | |
| SH_LINKF (Selection Marker + HA2) | |
| HR_R | |
| dNTP (dNTP mixture 2.5mM TaKaRa) | |
| Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE) | |
| 10x buffer (10x cloned Pfu Reaction buffer) | |
| DW (3rd sterile water) | |
| 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’ | 43bp |
| Materials | Volume |
| 5’UTR - Fusion Antibody receptor (template)
| |
| HA1 - Selection Marker (template)
| |
| HR_F (for homologous recombinant) | |
| SH_LINKR | |
| dNTP (dNTP mixture 2.5mM TaKaRa) | |
| Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE) | |
| 10x buffer (10x cloned Pfu Reaction buffer) | |
| DW (3rd sterile water) | |
| 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 | Volume |
| HA2 + 3'UTR (template)
| |
| 5’UTR - Fusion Antibody receptor - HA1 - Selection Marker (template)
| |
| HR_F (for homologous recombinant) | |
| HR_R | |
| dNTP (dNTP mixture 2.5mM TaKaRa) | |
| Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE) | |
| 10x buffer (10x cloned Pfu Reaction buffer) | |
| DW (3rd sterile water) | |
| Total | 50ul |
September next
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