Team:KAIST-Korea/Project/Methods/PCR

From 2010.igem.org

(Difference between revisions)
(Proximal promoter (expression))
Line 901: Line 901:
<tr align=center>
<tr align=center>
<td>PC_LINKR</td><td>50pmole</td>
<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>
<tr align=center>
<tr align=center>

Revision as of 18:16, 16 September 2010

 

PCR Strategy

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


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




Ig-like (Submission)

PCR FGFR.jpg


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


MaterialsAmount
Fusion antibody receptor T vector (template)150pmole
IG_F50pmole
IG_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 + Ig-like (Submission)

PCR FGFR.jpg


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


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




Antibody (Submission)

PCR FGFR.jpg


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


MaterialsAmount
Fusion antibody receptor T vector (template)150pmole
Ab_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




V_Fusion Antibody Receptor (Expression)

PCR FGFR.jpg


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


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




B_FGFR1 (Submission)

PCR FGFR.jpg


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


MaterialsAmount
FGFR1(KRIBB) (template)150pmole
B_FGFR_F50pmole
B_FGFR_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




VB_FGFR1 (Expression)

PCR FGFR.jpg


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


MaterialsAmount
FGFR1(KRIBB) (template)150pmole
Linker_F50pmole
B_FGFR_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 (Expression)

PCR FGFR.jpg


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


MaterialsAmount
V_Fusion antibody receptor (template)150pmole
VB_FGFR1(2+3) (template)150pmole
SIG_F50pmole
B_FGFR_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 (expression)

PCR HR.jpg


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


MaterialsAmount
S.pombe genomic DNA (template)150pmole
HR_F50pmole
UF_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




3’UTR (expression)

PCR HR.jpg


PrimerDirectionSequencesLength
HU_LINKF (HA2 + 3’UTR)Forward5’-CCATATGACCCAGATTACGCTTGGAAATTAAATCGTGCGTAAG-3’43(21+22)bp
HR_RReverse5’ – GCGTTCTTTTTTAATTCTGAATTAA – 3’25bp


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




Selection Marker (expression)

PCR HR.jpg


PrimerDirectionSequencesLength
HS_LINKF (HA1 + Selection Marker)Forward5’-CCATATGACCCAGATTACGCTAGCAAGCTTAAGCTTAGCTAC-3’42(21+21)bp
SH_LINKRReverse5’-GTCAGGAACATCGTATGGGTAGACCTGCAGAAGCTTAAGCTTG-3’43(21+22)bp


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




Core promoter + GFP (expression)

PCR GAS.jpg


PrimerDirectionSequencesLength
PC_LINKF (Proximal + Core)Forward5’–CAGAATAGACACACGGGCCGACATTGAAGATATATAAAGGAAG–3’43(20+23)bp
CGFP_R (Core promoter + GFP)Reverse3’-CGACATATGCTTGTACAGCTCG-5’22bp


MaterialsAmount
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
Total50ul

PCR cycle.jpg




Proximal promoter (expression)

PCR GAS.jpg


PrimerDirectionSequencesLength
PP_F (Proximal promoter)Forward5’-CGCCTGCAGGCGAATAGCTGG–3’21bp
PC_LINKRReverse5’–CTTCCTTTATATATCTTCAATGTCGGCCCGTGTGTCTATTCTG–3’43(23+20)bp


MaterialsAmount
Proximal promoter T vector (template)150pmole
PP_F (Proximal promoter)50pmole
PC_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




GAS Promoter (expression)

PCR GAS.jpg


PrimerDirectionSequencesLength
PP_F (Proximal promoter)Forward5’-CGCCTGCAGGCGAATAGCTGG–3’21bp
CGFP_R (Core promoter + GFP)Reverse3’-CGACATATGCTTGTACAGCTCG-5’22bp


MaterialsAmount
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
Total50ul

PCR cycle.jpg




HA1 (expression)

PCR HR.jpg


PrimerDirectionSequencesLength
FH_LINKF (FGFR + HA1)Forward5’-GCGGACTCAAACGCCGCTGATACCCATACGATGTTCCTGAC-3’41(20+21)bp
HS_LINKRReverse5’-GTAGCTAAGCTTAAGCTTGCTAGCGTAATCTGGGTCATATGG-3’42(21+21)bp


MaterialsAmount
HA T vector (template)150pmole
FH_LINKF (FGFR + HA1)50pmole
HS_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 (expression)

PCR HR.jpg


PrimerDirectionSequencesLength
SH_LINKF (Selection Marker + HA2)Forward5’-CAAGCTTAAGCTTCTGCAGGTCTACCCATACGATGTTCCTGAC-3’43(22+21)bp
HU_LINKRReverse5’-CTTACGCACGATTTAATTTCCAAGCGTAATCTGGGTCATATGG-3’43(22+21)bp


MaterialsAmount
HA T vector (template)150pmole
SH_LINKF (Selection Marker + HA2)50pmole
HU_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




Fusion antibody receptor (expression)

PCR HR.jpg


PrimerDirectionSequencesLength
UF_LinkF (5’UTR+FGFR Linker)Forward5’-GTAAAAAAGAAAAATGTTGTTTATGGTCTTTTTAAATTCCTCTC-3’44(22+22)bp
FH_LINKRReverse5’-GTCAGGAACATCGTATGGGTATCAGCGGCGTTTGAGTCCGC-3’41(21+20)bp


MaterialsAmount
Fusion Antibody receptor (template)150pmole
UF_LinkF (5’UTR+FGFR Linker)50pmole
FH_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




5’UTR + Fusion Antibody receptor (expression)

PCR HR.jpg


PrimerDirectionSequencesLength
HR_F (for homologous recombinant)Forward5’-AAACCAGTTTTGCCAAAACAAC-3’22bp
FH_LINKRReverse5’-GTCAGGAACATCGTATGGGTATCAGCGGCGTTTGAGTCCGC-3’41(21+20)bp


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-3’41(20+21)bp
SH_LINKRReverse5’-GTCAGGAACATCGTATGGGTAGACCTGCAGAAGCTTAAGCTTG-3’43(21+22)bp


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-3’43(22+21)bp
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-3’43(21+22)bp


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