Team:KAIST-Korea/Notebook/Diary/September

From 2010.igem.org

(Difference between revisions)

Revision as of 15:27, 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	Amount
Fusion antibody receptor T vector (template)	150pmole
SIG_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

Antibody (Submission)

Primer	Direction	Sequences	Length
Ab_F	Forward	5’-ACCCAGTCTCCAGCAATCATGTC-3’	23bp
Ab_R	Reverse	5’-GGGGCTGTTGTTTTGGCTGAGG-3’	22bp

Materials	Amount
Fusion antibody receptor T vector (template)	150pmole
Ab_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

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	Amount
Fusion antibody receptor T vector (template)	150pmole
SIG_F	50pmole
Linker_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

B_FGFR1 (Submission)

Primer	Direction	Sequences	Length
B_FGFR_F	Forward	5’-GTTCTGGAAGCCCTGGAAGAGAG-3’	23bp
B_FGFR_R	Reverse	5’-TCAGCGGCGTTTGAGTCCGCCAT-3’	23bp

Materials	Amount
FGFR1(KRIBB) (template)	150pmole
B_FGFR_F	50pmole
B_FGFR_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

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	Amount
FGFR1(KRIBB) (template)	150pmole
Linker_F	50pmole
B_FGFR_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 (Expression)

Primer	Direction	Sequences	Length
SIG_F	Forward	5’-ATGGTCTTTTTAAATTCCTCTCCC-3’	24bp
B_FGFR_R	Reverse	5’-TCAGCGGCGTTTGAGTCCGCCAT-3’	23bp

Materials	Amount
V_Fusion antibody receptor (template)	150pmole
VB_FGFR1(2+3) (template)	150pmole
SIG_F	50pmole
B_FGFR_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 (expression)

Primer	Direction	Sequences	Length
HR_F	Forward	5’-AAACCAGTTTTGCCAAAACAAC-3’	22bp
UF_LinkR	Reverse	5’ –GAGAGGAATTTAAAAAGACCATAAACAACATTTTTCTTTTTTAC - 3’	44(22+22)bp

Materials	Amount
S.pombe genomic DNA (template)	150pmole
HR_F	50pmole
UF_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

3’UTR (expression)

Primer	Direction	Sequences	Length
HU_LINKF (HA2 + 3’UTR)	Forward	5’-CCATATGACCCAGATTACGCTTGGAAATTAAATCGTGCGTAAG-3’	43(21+22)bp
HR_R	Reverse	5’ – GCGTTCTTTTTTAATTCTGAATTAA – 3’	25bp

Materials	Amount
S.pombe genomic DNA (template)	150pmole
HU_LINKF (HA2 + 3’UTR)	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

Selection Marker (expression)

Primer	Direction	Sequences	Length
HS_LINKF (HA1 + Selection Marker)	Forward	5’-CCATATGACCCAGATTACGCTAGCAAGCTTAAGCTTAGCTAC-3’	42(21+21)bp
SH_LINKR	Reverse	5’-GTCAGGAACATCGTATGGGTAGACCTGCAGAAGCTTAAGCTTG-3’	43(21+22)bp

Materials	Amount
pREP42-GFP vector (template)	150pmole
HS_LINKF (HA1 + Selection Marker)	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

Core promoter + GFP (expression)

Primer	Direction	Sequences	Length
PC_LINKF (Proximal + Core)	Forward	5’–CAGAATAGACACACGGGCCGACATTGAAGATATATAAAGGAAG–3’	43(20+23)bp
CGFP_R (Core promoter + GFP)	Reverse	3’-CGACATATGCTTGTACAGCTCG-5’	22bp

Materials	Amount
pREP42-GFP vector (template)	150pmole
PC_LINKF (Proximal + Core)	50pmole
CGFP_R (Core promoter + GFP)	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

Proximal promoter (expression)

Primer	Direction	Sequences	Length
PP_F (Proximal promoter)	Forward	5’-CGCCTGCAGGCGAATAGCTGG–3’	21bp
PC_LINKR	Reverse	5’–CTTCCTTTATATATCTTCAATGTCGGCCCGTGTGTCTATTCTG–3’	43(23+20)bp

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

@@ Line 940: / Line 940: @@
 </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>
+<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>21bp</td>
 </tr>
 <tr align=center>
-<td>PC_LINKR</td><td>Reverse</td><td>5’–CTTCCTTTATATATCTTCAATGTCGGCCCGTGTGTCTATTCTG–3’</td><td>43bp</td>
+<td>PC_LINKR</td><td>Reverse</td><td>5’–CTTCCTTTATATATCTTCAATGTCGGCCCGTGTGTCTATTCTG–3’</td><td>43(23+20)bp</td>
 </tr>
 </table>