Difference between revisions of "Team:Vilnius-Lithuania/Notebook"

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<a href='https://2016.igem.org/Team:Vilnius-Lithuania/Safety'>Safety</a>
 
<a href='https://2016.igem.org/Team:Vilnius-Lithuania/Safety'>Safety</a>
 
</div>
 
</div>
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<h2 class="Raleway" style="text-align:center;"> 09.03 </h2>
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<p class="Raleway"> Transformation of pET-Duet pET-Duet N-terminus Strep-tag C-terminus His-tag in DH5alpha</p>
 +
<h2 class="Raleway" style="text-align:center;">09.05</h2>
 +
 +
<p class="Raleway"> Selection of corrent colonies and inoculation in minipreps for DNA extraction.
 +
</p>
 +
 +
<p class="Raleway">1. Miniprep extraction
 +
• Extraction of pET-Duet pET-Duet N-terminus Strep-tag C-terminus His-tag. Concentration 30 ng/µl;
 +
• Need to cut  ̴ 10 µg * with FD NcoI and FD XhoI
 +
Concentation = 7,8 ng/µl
 +
 +
2. psB1C3
 +
Picked 5 – 8 pSB1C3 and ran an agarose gel:
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3. Ligation with T7 polymerase</p>
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 +
<h2 class="Raleway" style="text-align:center;">09.06</h2>
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 +
<p class="Raleway"> Prepare and autoclave growth medium: 20ml x2.
 +
</p>
 +
 +
<h2 class="Raleway" style="text-align:center;">09.08</h2>
 +
 +
<p class="Raleway"> Minipreps purification (RFP + pSB1C3). Bacteria are pink in liquid medium. Centrifuged 10ml (in 15ml tubes).
 +
Prepared 9 RFP (1) minipreps for tomorows purification. Antibiotics Cm.
 +
We got oligonucleotides: channels (PheP), tRNR.
 +
Made PCR with a temperature gradient for PheP, tRNR biobrick, DH10B tRNR, cProm tRNR.
 +
Polymerase: Phusion.
 +
</p>
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 +
<h2 class="Raleway" style="text-align:center;">09.09</h2>
 +
 +
<p class="Raleway"> Minipreps purification.
 +
Yesterday’s PCR succeeded.
 +
Purification with PCR purification kit.
 +
pSB1C3 + RFP restriction.
 +
RE: EamII05I, NcoI, EcoRI, PstI.
 +
Fragments purificated through column.
 +
Transformed terminal sequence (biobrick) from kit plate 3 (3F). → DH10B
 +
Insert and vector ligation.
 +
Vector: pSB1C3 Insert: PheP, tRNA B, tRNA D, tRNA C. Transform into DH10B.
 +
</p>
 +
 +
<h2 class="Raleway" style="text-align:center;">09.10</h2>
 +
 +
<p class="Raleway"> Yesterday’s transformation and PheP cloning did not succeed.
 +
Colony PCR.
 +
PheP and terminator transformation. Competent cells DH5α.
 +
pSB1C3 + RFP miniprep purification.
 +
Restriction with FD EamII05I, FD NcoI, FD EcoRI, FD PstI
 +
Fragments cutting: PheP and tRNA D with FD EcoRI and FD PstI
 +
Fragments and vectors after RE purification through column
 +
Ligation. PheP into pSB1C3, tRNA with promoter DH10B into pSB1C3
 +
PheP, tRNA D and terminator transformation.
 +
Colony PCR did not succeed. Colonies are red that means RFP remained.
 +
</p>
 +
 +
<h2 class="Raleway" style="text-align:center;">09.11</h2>
 +
 +
<p class="Raleway"> Yesterday’s transformation did not succeed. Today we are repeating experiment with other RE.
 +
Vector: pSB1C3; RE: FD EamII05I, FD PstAI, FD EcoRI, FD PstI.
 +
</p>
 +
 +
<h2 class="Raleway" style="text-align:center;">09.12</h2>
 +
 +
<p class="Raleway"> Miniprep DNA extraction (pSB1C3+RFP)
 +
Digest with EcoRI and PstI
 +
Colonies PCR
 +
Arrived new genes: modified gp45 is csm4. Preparation for cloning with PCR. Phusion polymerase.
 +
</p>
 +
 +
<h2 class="Raleway" style="text-align:center;">09.13</h2>
 +
 +
<p class="Raleway"> Miniprep DNA extraction: YFP, RFP, PheP, tRNAB, tRNAD, tRNAC, TERM.
 +
Digestion of pSB1C3. Ligation
 +
Transformation pSB1C3+tRNAB, pSB1C3+tRNAD, pSB1C3+tRNAC, pSB1C3+PheP.
 +
PCR of GP45 and Csm4. Purification with PCR purification kit.
 +
</p>
 +
 +
<h2 class="Raleway" style="text-align:center;">09.14</h2>
 +
 +
<p class="Raleway"> Preparation of minipreps. Extraction of DNA from minipreps with TERM plasmids. Ter2 digested with FD EcoRI and FD PstI.
 +
</p>
  
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<p class="Raleway">Text here</p>
 
 
</div>
 
</div>
 
</div>
 
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Revision as of 02:20, 20 October 2016

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Notebook

09.03

Transformation of pET-Duet pET-Duet N-terminus Strep-tag C-terminus His-tag in DH5alpha

09.05

Selection of corrent colonies and inoculation in minipreps for DNA extraction.

1. Miniprep extraction • Extraction of pET-Duet pET-Duet N-terminus Strep-tag C-terminus His-tag. Concentration 30 ng/µl; • Need to cut ̴ 10 µg * with FD NcoI and FD XhoI Concentation = 7,8 ng/µl 2. psB1C3 Picked 5 – 8 pSB1C3 and ran an agarose gel: 3. Ligation with T7 polymerase

09.06

Prepare and autoclave growth medium: 20ml x2.

09.08

Minipreps purification (RFP + pSB1C3). Bacteria are pink in liquid medium. Centrifuged 10ml (in 15ml tubes). Prepared 9 RFP (1) minipreps for tomorows purification. Antibiotics Cm. We got oligonucleotides: channels (PheP), tRNR. Made PCR with a temperature gradient for PheP, tRNR biobrick, DH10B tRNR, cProm tRNR. Polymerase: Phusion.

09.09

Minipreps purification. Yesterday’s PCR succeeded. Purification with PCR purification kit. pSB1C3 + RFP restriction. RE: EamII05I, NcoI, EcoRI, PstI. Fragments purificated through column. Transformed terminal sequence (biobrick) from kit plate 3 (3F). → DH10B Insert and vector ligation. Vector: pSB1C3 Insert: PheP, tRNA B, tRNA D, tRNA C. Transform into DH10B.

09.10

Yesterday’s transformation and PheP cloning did not succeed. Colony PCR. PheP and terminator transformation. Competent cells DH5α. pSB1C3 + RFP miniprep purification. Restriction with FD EamII05I, FD NcoI, FD EcoRI, FD PstI Fragments cutting: PheP and tRNA D with FD EcoRI and FD PstI Fragments and vectors after RE purification through column Ligation. PheP into pSB1C3, tRNA with promoter DH10B into pSB1C3 PheP, tRNA D and terminator transformation. Colony PCR did not succeed. Colonies are red that means RFP remained.

09.11

Yesterday’s transformation did not succeed. Today we are repeating experiment with other RE. Vector: pSB1C3; RE: FD EamII05I, FD PstAI, FD EcoRI, FD PstI.

09.12

Miniprep DNA extraction (pSB1C3+RFP) Digest with EcoRI and PstI Colonies PCR Arrived new genes: modified gp45 is csm4. Preparation for cloning with PCR. Phusion polymerase.

09.13

Miniprep DNA extraction: YFP, RFP, PheP, tRNAB, tRNAD, tRNAC, TERM. Digestion of pSB1C3. Ligation Transformation pSB1C3+tRNAB, pSB1C3+tRNAD, pSB1C3+tRNAC, pSB1C3+PheP. PCR of GP45 and Csm4. Purification with PCR purification kit.

09.14

Preparation of minipreps. Extraction of DNA from minipreps with TERM plasmids. Ter2 digested with FD EcoRI and FD PstI.