From 2010.igem.org
(Difference between revisions)
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| <table border=1> | | <table border=1> |
| <tr align=center> | | <tr align=center> |
- | <td><b>Materials</b></td><td><b>Volume</b></td> | + | <td><b>Materials</b></td><td><b>Amount</b></td> |
| </tr> | | </tr> |
| <tr align=center> | | <tr align=center> |
- | <td>Fusion antibody receptor T vector (template)</td><td></td> | + | <td>Fusion antibody receptor T vector (template)</td><td>150pmole</td> |
| </tr> | | </tr> |
| <tr align=center> | | <tr align=center> |
- | <td>IG_F</td><td></td> | + | <td>IG_F</td><td>50pmole</td> |
| </tr> | | </tr> |
| <tr align=center> | | <tr align=center> |
- | <td>IG_R</td><td></td> | + | <td>IG_R</td><td>50pmole</td> |
| </tr> | | </tr> |
| <tr align=center> | | <tr align=center> |
- | <td>dNTP (dNTP mixture 2.5mM TaKaRa)</td><td></td> | + | <td>dNTP (dNTP mixture 2.5mM TaKaRa)</td><td>5ul</td> |
| </tr> | | </tr> |
| <tr align=center> | | <tr align=center> |
- | <td>Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE)</td><td></td> | + | <td>Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE)</td><td>0.25ul</td> |
| </tr> | | </tr> |
| <tr align=center> | | <tr align=center> |
- | <td>10x buffer (10x cloned Pfu Reaction buffer)</td><td></td> | + | <td>10x buffer (10x cloned Pfu Reaction buffer)</td><td>5ul</td> |
| </tr> | | </tr> |
| <tr align=center> | | <tr align=center> |
- | <td>DW (3rd sterile water)</td><td></td> | + | <td>DW (3rd sterile water)</td><td>rest</td> |
| </tr> | | </tr> |
| <tr align=center> | | <tr align=center> |
Revision as of 15:19, 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 | 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 | 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)
Primer | Direction | Sequences | Length |
SIG_F | Forward | 5’-ATGGTCTTTTTAAATTCCTCTCCC-3’ | 24bp |
B_FGFR_R | Reverse | 5’-TCAGCGGCGTTTGAGTCCGCCAT-3’ | 23bp |
Materials | Volume |
V_Fusion antibody receptor (template) | |
VB_FGFR1(2+3) (template) | |
SIG_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 |
5’UTR (expression)
Primer | Direction | Sequences | Length |
HR_F | Forward | 5’-AAACCAGTTTTGCCAAAACAAC-3’ | 22bp |
UF_LinkR | Reverse | 5’ –GAGAGGAATTTAAAAAGACCATAAACAACATTTTTCTTTTTTAC - 3’ | 44bp |
Materials | Volume |
S.pombe genomic DNA (template) | |
HR_F | |
UF_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 |
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 | 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’ | 41bp |
HS_LINKR | Reverse | 5’-GTAGCTAAGCTTAAGCTTGCTAGCGTAATCTGGGTCATATGG-3’ | 42bp |
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’ | 43bp |
HU_LINKR | Reverse | 5’-CTTACGCACGATTTAATTTCCAAGCGTAATCTGGGTCATATGG-3’ | 43bp |
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’ | 44bp |
FH_LINKR | Reverse | 5’-GTCAGGAACATCGTATGGGTATCAGCGGCGTTTGAGTCCGC-3’ | 41bp |
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’ | 41bp |
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’ | 41bp |
SH_LINKR | Reverse | 5’-GTCAGGAACATCGTATGGGTAGACCTGCAGAAGCTTAAGCTTG-3’ | 43bp |
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’ | 43bp |
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’ | 43bp |
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 next
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