Team:KAIST-Korea/Notebook/Diary/September

From 2010.igem.org

(Difference between revisions)
(HA1 + Selection Marker (expression))
(5’UTR + Fusion Antibody receptor (expression))
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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>5'UTR (template)</td><td></td>
+
<td>5'UTR (template)</td><td>150pmole</td>
</tr>
</tr>
<tr align=center>
<tr align=center>
-
<td>Fusion Antibody receptor (template)</td><td></td>
+
<td>Fusion Antibody receptor (template)</td><td>150pmole</td>
</tr>
</tr>
<tr align=center>
<tr align=center>
-
<td>FH_LINKF (FGFR + HA1)</td><td></td>
+
<td>FH_LINKF (FGFR + HA1)</td><td>50pmole</td>
</tr>
</tr>
<tr align=center>
<tr align=center>
-
<td>SH_LINKR</td><td></td>
+
<td>SH_LINKR</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 15:16, 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

  1. 500mL LB broth + agar 1.5% (7.5g)
  2. Do auto clave
  3. Cool it down until it reaches 50~60℃
  4. Add ampicillin(1000x 100mg/mL) and stir
  5. putting out bubbles on culture media
  6. pouling on petri dish and wait for 1 hour

Culture

  1. Competent cell + DN10A and leave it in ice for 30min
  2. Do heat shock for 45 sec and store it in ice for 2min
  3. Stabilize it in incubator for 30 to 40min
  4. Do spreading


September ?

V_STAT1 (expression)

PCR STAT1.jpg


PrimerDirectionSequencesLength
V_STAT_FForward(NdeI)5’-TGTTCATATGGCTAATGTCTCAGTGGTACGAACTTCAG-3’24bp
V_STAT_RReverse(BamHI)5’-TATCGGATCCGAATTTACACTTCAGACACAGAAATCAAC-3’25bp


MaterialsAmount
STAT1(KRIBB) (template)150pmole
V_STAT_F50pmole
V_STAT_R50pmole
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
Total50ul

PCR cycle.jpg




STAT1 (submission)

PCR STAT1.jpg


PrimerDirectionSequencesLength
STAT_FForward5’-ATGTCTCAGTGGTACGAACTTCAG-3’24bp
STAT_RReverse5’-TTACACTTCAGACACAGAAATCAAC-3’25bp


MaterialsAmount
STAT1(KRIBB) (template)150pmole
STAT_F50pmole
STAT_R50pmole
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
Total50ul

PCR cycle.jpg




Fusion Antibody Receptor (Submission)

PCR FGFR.jpg


PrimerDirectionSequencesLength
SIG_FForward5’-ATGGTCTTTTTAAATTCCTCTCCC-3’24bp
Ab_RReverse5’-GGGGCTGTTGTTTTGGCTGAGG-3’22bp


MaterialsAmount
Fusion antibody receptor T vector (template)150pmole
SIG_F50pmole
Ab_R50pmole
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
Total50ul

PCR cycle.jpg




Signal Peptide (Submission)

PCR FGFR.jpg


PrimerDirectionSequencesLength
SIG_FForward5’-ATGGTCTTTTTAAATTCCTCTCCC-3’24bp
SIG_RReverse5’-AGCCGCCACCAACCGAGTAGAAA-3’23bp


MaterialsVolume
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)
Total50ul

PCR cycle.jpg




Ig-like (Submission)

PCR FGFR.jpg


PrimerDirectionSequencesLength
IG_FForward5’-AGGCCGTCCCCGACCTTGCCTG-3’22bp
IG_RReverse5’-GGAGTCATCATCATCATCATCATC-3’24bp


MaterialsVolume
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)
Total50ul

PCR cycle.jpg




Signal Peptide + Ig-like (Submission)

PCR FGFR.jpg


PrimerDirectionSequencesLength
SIG_FForward5’-ATGGTCTTTTTAAATTCCTCTCCC-3’24bp
IG_RReverse5’-GGAGTCATCATCATCATCATCATC-3’24bp


MaterialsVolume
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)
Total50ul

PCR cycle.jpg




Antibody (Submission)

PCR FGFR.jpg


PrimerDirectionSequencesLength
Ab_FForward5’-ACCCAGTCTCCAGCAATCATGTC-3’23bp
Ab_RReverse5’-GGGGCTGTTGTTTTGGCTGAGG-3’22bp


MaterialsVolume
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)
Total50ul

PCR cycle.jpg




V_Fusion Antibody Receptor (Expression)

PCR FGFR.jpg


PrimerDirectionSequencesLength
SIG_FForward5’-ATGGTCTTTTTAAATTCCTCTCCC-3’24bp
Linker_RReverse5’-CTCTCTTCCAGGGCTTCCAGAACGGGGCTGTTGTTTTGGCTGAGGA-3’46(23+23)bp


MaterialsVolume
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)
Total50ul

PCR cycle.jpg




B_FGFR1 (Submission)

PCR FGFR.jpg


PrimerDirectionSequencesLength
B_FGFR_FForward5’-GTTCTGGAAGCCCTGGAAGAGAG-3’23bp
B_FGFR_RReverse5’-TCAGCGGCGTTTGAGTCCGCCAT-3’23bp


MaterialsVolume
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)
Total50ul

PCR cycle.jpg




VB_FGFR1 (Expression)

PCR FGFR.jpg


PrimerDirectionSequencesLength
Linker_FForward5’-TCCTCAGCCAAAACAACAGCCCCGTTCTGGAAGCCCTGGAAGAGAG-3’46(23+23)bp
B_FGFR_RReverse5’-TCAGCGGCGTTTGAGTCCGCCAT-3’23bp


MaterialsVolume
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)
Total50ul

PCR cycle.jpg




Fusion Antibody Receptor (Expression)

PCR FGFR.jpg


PrimerDirectionSequencesLength
SIG_FForward5’-ATGGTCTTTTTAAATTCCTCTCCC-3’24bp
B_FGFR_RReverse5’-TCAGCGGCGTTTGAGTCCGCCAT-3’23bp


MaterialsVolume
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)
Total50ul

PCR cycle.jpg




5’UTR (expression)

PCR HR.jpg


PrimerDirectionSequencesLength
HR_FForward5’-AAACCAGTTTTGCCAAAACAAC-3’22bp
UF_LinkRReverse5’ –GAGAGGAATTTAAAAAGACCATAAACAACATTTTTCTTTTTTAC - 3’44bp


MaterialsVolume
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)
Total50ul

PCR cycle.jpg




3’UTR (expression)

PCR HR.jpg


PrimerDirectionSequencesLength
HU_LINKF (HA2 + 3’UTR)Forward5’-CCATATGACCCAGATTACGCTTGGAAATTAAATCGTGCGTAAG-3’43bp
HR_RReverse5’ – GCGTTCTTTTTTAATTCTGAATTAA – 3’25bp


MaterialsVolume
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)
Total50ul

PCR cycle.jpg




Selection Marker (expression)

PCR GAS.jpg


PrimerDirectionSequencesLength
HS_LINKF (HA1 + Selection Marker)Forward5’-CCATATGACCCAGATTACGCTAGCAAGCTTAAGCTTAGCTAC-3’42bp
SH_LINKRReverse5’-GTCAGGAACATCGTATGGGTAGACCTGCAGAAGCTTAAGCTTG-3’43bp


MaterialsVolume
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)
Total50ul

PCR cycle.jpg




Core promoter + GFP (expression)

PCR GAS.jpg


PrimerDirectionSequencesLength
PC_LINKF (Proximal + Core)Forward5’–CAGAATAGACACACGGGCCGACATTGAAGATATATAAAGGAAG–3’43bp
CGFP_R (Core promoter + GFP)Reverse3’-CGACATATGCTTGTACAGCTCG-5’22bp


MaterialsVolume
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)
Total50ul

PCR cycle.jpg




Proximal promoter (expression)

PCR GAS.jpg


PrimerDirectionSequencesLength
PP_F (Proximal promoter)Forward5’-CGCCTGCAGGCGAATAGCTGG–3’41bp
PC_LINKRReverse5’–CTTCCTTTATATATCTTCAATGTCGGCCCGTGTGTCTATTCTG–3’43bp


MaterialsVolume
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)
Total50ul

PCR cycle.jpg




HA1 (expression)

PCR HR.jpg


PrimerDirectionSequencesLength
FH_LINKF (FGFR + HA1)Forward5’-GCGGACTCAAACGCCGCTGATACCCATACGATGTTCCTGAC-3’41bp
HS_LINKRReverse5’-GTAGCTAAGCTTAAGCTTGCTAGCGTAATCTGGGTCATATGG-3’42bp


MaterialsVolume
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)
Total50ul

PCR cycle.jpg




HA2 (expression)

PCR HR.jpg


PrimerDirectionSequencesLength
SH_LINKF (Selection Marker + HA2)Forward5’-CAAGCTTAAGCTTCTGCAGGTCTACCCATACGATGTTCCTGAC-3’43bp
HU_LINKRReverse5’-CTTACGCACGATTTAATTTCCAAGCGTAATCTGGGTCATATGG-3’43bp


MaterialsVolume
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)
Total50ul

PCR cycle.jpg




Fusion antibody receptor (expression)

PCR HR.jpg


PrimerDirectionSequencesLength
UF_LinkF (5’UTR+FGFR Linker)Forward5’-GTAAAAAAGAAAAATGTTGTTTATGGTCTTTTTAAATTCCTCTC-3’44bp
FH_LINKRReverse5’-GTCAGGAACATCGTATGGGTATCAGCGGCGTTTGAGTCCGC-3’41bp


MaterialsVolume
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)
Total50ul

PCR cycle.jpg




5’UTR + Fusion Antibody receptor (expression)

PCR HR.jpg


PrimerDirectionSequencesLength
HR_F (for homologous recombinant)Forward5’-AAACCAGTTTTGCCAAAACAAC-3’22bp
FH_LINKRReverse5’-GTCAGGAACATCGTATGGGTATCAGCGGCGTTTGAGTCCGC-3’41bp


MaterialsAmount
5'UTR (template)150pmole
Fusion Antibody receptor (template)150pmole
FH_LINKF (FGFR + HA1)50pmole
SH_LINKR50pmole
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
Total50ul

PCR cycle.jpg




HA1 + Selection Marker (expression)

PCR HR.jpg


PrimerDirectionSequencesLength
FH_LINKF (FGFR + HA1)Forward5’-GCGGACTCAAACGCCGCTGATACCCATACGATGTTCCTGAC-3’41bp
SH_LINKRReverse5’-GTCAGGAACATCGTATGGGTAGACCTGCAGAAGCTTAAGCTTG-3’43bp


MaterialsAmount
HA1 (template)150pmole
Selection Marker (template)150pmole
FH_LINKF (FGFR + HA1)50pmole
SH_LINKR50pmole
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
Total50ul

PCR cycle.jpg




HA2 + 3'UTR (expression)

PCR HR.jpg


PrimerDirectionSequencesLength
SH_LINKF (Selection Marker + HA2)Forward5’-CAAGCTTAAGCTTCTGCAGGTCTACCCATACGATGTTCCTGAC-3’43bp
HR_RReverse5’ – GCGTTCTTTTTTAATTCTGAATTAA – 3’25bp


MaterialsAmount
HA2 (template)150pmole
3’UTR (template)150pmole
SH_LINKF (Selection Marker + HA2)50pmole
HR_R50pmole
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
Total50ul

PCR cycle.jpg




5’UTR - Fusion Antibody receptor + HA1 - Selection Marker (expression)

PCR HR.jpg


PrimerDirectionSequencesLength
HR_F(for homologous recombinant)Forward5’-AAACCAGTTTTGCCAAAACAAC-3’22bp
SH_LINKRReverse5’-GTCAGGAACATCGTATGGGTAGACCTGCAGAAGCTTAAGCTTG-3’43bp


MaterialsAmount
5’UTR - Fusion Antibody receptor (template) 150pmole
HA1 - Selection Marker (template) 150pmole
HR_F (for homologous recombinant)50pmole
SH_LINKR50pmole
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
Total50ul

PCR cycle.jpg




Homologous Recombinant (expression)

PCR HR.jpg


PrimerDirectionSequencesLength
HR_F(for homologous recombinant)Forward5’-AAACCAGTTTTGCCAAAACAAC-3’22bp
HR_RReverse5’ – GCGTTCTTTTTTAATTCTGAATTAA – 3’25bp


MaterialsAmount
HA2 + 3'UTR (template) 150pmole
5’UTR - Fusion Antibody receptor - HA1 - Selection Marker (template) 150pmole
HR_F (for homologous recombinant)50pmole
HR_R50pmole
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
Total50ul

PCR cycle.jpg




September next