From 2010.igem.org
(Difference between revisions)
Line 589:
Line 589:
=== Selection Marker (expression) ===
=== Selection Marker (expression) ===
=== Core promoter + GFP (expression) ===
=== Core promoter + GFP (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>PC_LINKF (Proximal + Core)</td><td>Forward</td><td>5’–CAGAATAGACACACGGGCCGACATTGAAGATATATAAAGGAAG–3’</td><td>43bp</td>
+ </tr>
+ <tr align=center>
+ <td>CGFP_R (Core promoter + GFP)</td><td>Reverse</td><td>3’-<font color = blue>CGA</font><font color = red>CATATG</font><font color = blue>CTT</font>GTACAGCTCG-5’</td><td>22bp</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>pREP42-GFP vector (template)</td><td></td>
+ </tr>
+ <tr align=center>
+ <td>PC_LINKF (Proximal + Core)</td><td></td>
+ </tr>
+ <tr align=center>
+ <td>CGFP_R (Core promoter + GFP)</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>
+
=== Proximal promoter (expression) ===
=== Proximal promoter (expression) ===
Revision as of 15:01, 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’-TGTT CATATG GCTA ATGTCTCAGTGGTACGAACTTCAG-3’ 24bp
V_STAT_R Reverse(BamHI) 5’-TATC GGATCC GAAT TTACACTTCAGACACAGAAATCAAC-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)
Fusion Antibody Receptor (Expression)
5’UTR (expression)
3’UTR (expression)
Selection Marker (expression)
Core promoter + GFP (expression)
Primer Direction Sequences Length
PC_LINKF (Proximal + Core) Forward 5’–CAGAATAGACACACGGGCCGACATTGAAGATATATAAAGGAAG–3’ 43bp
CGFP_R (Core promoter + GFP) Reverse 3’-CGA CATATG CTT GTACAGCTCG-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’-CGC CTGCAG GCG AATAGCTGG–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