Team:KAIST-Korea/Project/Methods/PCR

From 2010.igem.org

(Difference between revisions)

Latest revision as of 07:08, 20 October 2010

PCR Strategy

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

GAS Promoter (expression)

Primer	Direction	Sequences	Length
PP_F (Proximal promoter)	Forward	5’-CGCCTGCAGGCGAATAGCTGG–3’	21bp
CGFP_R (Core promoter + GFP)	Reverse	3’-CGACATATGCTTGTACAGCTCG-5’	22bp

Materials	Amount
Proximal promoter(template)	150pmole
Core promoter + GFP(template)	150pmole
PP_F (Proximal promoter)	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

HA1 (expression)

Primer	Direction	Sequences	Length
FH_LINKF (FGFR + HA1)	Forward	5’-GCGGACTCAAACGCCGCTGATACCCATACGATGTTCCTGAC-3’	41(20+21)bp
HS_LINKR	Reverse	5’-GTAGCTAAGCTTAAGCTTGCTAGCGTAATCTGGGTCATATGG-3’	42(21+21)bp

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’	43(22+21)bp
HU_LINKR	Reverse	5’-CTTACGCACGATTTAATTTCCAAGCGTAATCTGGGTCATATGG-3’	43(22+21)bp

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’	44(22+22)bp
FH_LINKR	Reverse	5’-GTCAGGAACATCGTATGGGTATCAGCGGCGTTTGAGTCCGC-3’	41(21+20)bp

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’	41(21+20)bp

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’	41(20+21)bp
SH_LINKR	Reverse	5’-GTCAGGAACATCGTATGGGTAGACCTGCAGAAGCTTAAGCTTG-3’	43(21+22)bp

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’	43(22+21)bp
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’	43(21+22)bp

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 6: / Line 6: @@
 <td valign="top">
+<html>
+<center>
+<a href="https://2010.igem.org/Team:KAIST-Korea/Project/Methods"><img src="https://static.igem.org/mediawiki/2010/6/6f/Method_button.png" width = 300></a>
+<a href="https://2010.igem.org/Team:KAIST-Korea/Project/Methods/PCR"><img src="https://static.igem.org/mediawiki/2010/e/e2/PCR-KAIST3.png" width = 300></a>
+<a href="https://2010.igem.org/Team:KAIST-Korea/Project/Methods/PCR_Result"><img src="https://static.igem.org/mediawiki/2010/9/94/PCR-Exp.png" width = 300></a></center>
+</html>
+<br>
+<br>
 <span style=font-size:30px><font face="Maiandra GD"><b>PCR Strategy</b></font></span>
@@ Line 13: / Line 21: @@
 <center>
-[[Image:PCR_STAT1.jpg]]
+[[Image:PCR_STAT1_m.jpg]]
 <br>
@@ Line 70: / Line 78: @@
 <center>
-[[Image:PCR_STAT1.jpg]]
+[[Image:PCR_STAT1_m.jpg]]
 <br>
@@ Line 901: / Line 909: @@
 <tr align=center>
 <td>PC_LINKR</td><td>50pmole</td>
+</tr>
+<tr align=center>
+<td>dNTP (dNTP mixture 2.5mM TaKaRa)</td><td>5ul</td>
+</tr>
+<tr align=center>
+<td>Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE)</td><td>0.25ul</td>
+</tr>
+<tr align=center>
+<td>10x buffer (10x cloned Pfu Reaction buffer)</td><td>5ul</td>
+</tr>
+<tr align=center>
+<td>DW (3rd sterile water)</td><td>rest</td>
+</tr>
+<tr align=center>
+<td>Total</td><td>50ul</td>
+</tr>
+</table>
+[[Image:PCR_cycle.jpg]]
+</center>
+<br>
+<br>
+<br>
+=== GAS Promoter (expression) ===
+<center>
+[[Image:PCR_GAS.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>21bp</td>
+</tr>
+<tr align=center>
+<td>CGFP_R (Core promoter + GFP)</td><td>Reverse</td><td>3’-CGACATATGCTTGTACAGCTCG-5’</td><td>22bp</td>
+</tr>
+</table>
+<br>
+<table border=1>
+<tr align=center>
+<td><b>Materials</b></td><td><b>Amount</b></td>
+</tr>
+<tr align=center>
+<td>Proximal promoter(template)</td><td>150pmole</td>
+</tr>
+<tr align=center>
+<td>Core promoter + GFP(template)</td><td>150pmole</td>
+</tr>
+<tr align=center>
+<td>PP_F (Proximal promoter)</td><td>50pmole</td>
+</tr>
+<tr align=center>
+<td>CGFP_R (Core promoter + GFP)</td><td>50pmole</td>
 </tr>
 <tr align=center>