Team:KAIST-Korea/Notebook/Diary/September

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Revision as of 14:58, 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)

Fusion Antibody Receptor (Expression)

5’UTR (expression)

3’UTR (expression)

Selection Marker (expression)

Core promoter + GFP (expression)

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

@@ Line 590: / Line 590: @@
 === Core promoter + GFP (expression) ===
 === Proximal promoter (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>PP_F (Proximal promoter)</td><td>Forward</td><td>5’-<font color = blue>CGC</font><font color = red>CTGCAG</font><font color = blue>GCG</font>AATAGCTGG–3’</td><td>41bp</td>
+</tr>
+<tr align=center>
+<td>PC_LINKR</td><td>Reverse</td><td>5’–CTTCCTTTATATATCTTCAATGTCGGCCCGTGTGTCTATTCTG–3’</td><td>43bp</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>Proximal promoter T vector (template)</td><td></td>
+</tr>
+<tr align=center>
+<td>PP_F (Proximal promoter)</td><td></td>
+</tr>
+<tr align=center>
+<td>PC_LINKR</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>
 === HA1 (expression) ===