Team:KAIST-Korea/Notebook/Diary/September

From 2010.igem.org

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(→Signal Peptide + Ig-like (Submission))
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=== Homologous Recombinant (expression) ===
=== Homologous Recombinant (expression) ===
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<center>
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[[Image:PCR_HR.jpg]]
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<br>
 +
 +
<table border=1>
 +
<tr align=center>
 +
<td>Primer</td><td>Direction</td><td>Sequences</td><td>Length</td>
 +
</tr>
 +
<tr align=center>
 +
<td>SIG_F</td><td>Forward</td><td>5’-ATGGTCTTTTTAAATTCCTCTCCC-3’</td><td>24bp</td>
 +
</tr>
 +
<tr align=center>
 +
<td>SIG_R</td><td>Reverse</td><td>5’-AGCCGCCACCAACCGAGTAGAAA-3’</td><td>23bp</td>
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</tr>
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</table>
 +
 +
<br>
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 +
<table border=1>
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<tr align=center>
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<td><b>Materials</b></td><td><b>Volume</b></td>
 +
</tr>
 +
<tr align=center>
 +
<td>HA2 + HR_R (template)<br>
 +
5’UTR - Fusion Antibody receptor + HA1 - Selection Marker (template)
 +
</td><td></td>
 +
</tr>
 +
<tr align=center>
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<td>SIG_F</td><td></td>
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</tr>
 +
<tr align=center>
 +
<td>SIG_R</td><td></td>
 +
</tr>
 +
<tr align=center>
 +
<td>dNTP (dNTP mixture 2.5mM TaKaRa)</td><td></td>
 +
</tr>
 +
<tr align=center>
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<td>Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE)</td><td></td>
 +
</tr>
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<tr align=center>
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<td>10x buffer (10x cloned Pfu Reaction buffer)</td><td></td>
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</tr>
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<tr align=center>
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<td>DW (3rd sterile water)</td><td></td>
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</tr>
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<tr align=center>
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<td>Total</td><td>50ul</td>
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</tr>
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</table>
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[[Image:PCR_cycle.jpg]]
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</center>

Revision as of 14:10, 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


MaterialsVolume
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)
Total50ul

PCR cycle.jpg


STAT1 (submission)

PCR STAT1.jpg


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


MaterialsVolume
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)
Total50ul

PCR cycle.jpg


Fusion Antibody Receptor (Submission)

PCR FGFR.jpg


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


MaterialsVolume
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)
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)

V_Fusion Antibody Receptor (Expression)

B_FGFR1 (Submission)

VB_FGFR1 (Expression)

Fusion Antibody Receptor (Expression)

5’UTR (expression)

3’UTR (expression)

Selection Marker (expression)

Core promoter + GFP (expression)

Proximal promoter (expression)

HA1 (expression)

HA2 (expression)

Fusion antibody receptor (expression)

5’UTR + Fusion Antibody receptor (expression)

HA1 + Selection Marker (expression)

HA2 + HR_R (expression)

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

Homologous Recombinant (expression)

PCR HR.jpg


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


MaterialsVolume
HA2 + HR_R (template)
5’UTR - Fusion Antibody receptor + HA1 - Selection Marker (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


PCR FGFR.jpg PCR HR.jpg PCR GAS.jpg

September next

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