Difference between revisions of "Team:SPSingapore/Notebook-Week-4"

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<table><tr><td>
 
<table><tr><td>
 
<div style = "border-top: 5px solid blue; width:750px; text-align:justify;margin-bottom: 20px; line-height:normal;">
 
<div style = "border-top: 5px solid blue; width:750px; text-align:justify;margin-bottom: 20px; line-height:normal;">
−
<h3 style ="margin-left:20px;">Week 4 (14/6 - 20/6)</h3>
+
<h3 style ="margin-left:20px;">Week 1 (24/5 - 30/5)</h3>
  
  
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<!----------------------- Entry Start ------------------------>
 
<!----------------------- Entry Start ------------------------>
 
<center>
 
<center>
−
<table style = "width:700px; border-collapse: collapse;">
+
<table style = "width:750px; border-collapse: collapse;">
−
 
+
 
<tr class = "tablenotebook"><td><p class = "paper">
 
<tr class = "tablenotebook"><td><p class = "paper">
−
14th June 2015
+
<u>May 25</u>
 
<br><br>
 
<br><br>
−
<strong>Yi Han</strong>
+
<strong>[Yi Han]</strong>
 
<br><br>
 
<br><br>
−
&bull; Miniprep of gfp plasmids, preparation of samples for sequencin
+
&bull; Received 2 bacterial stab cultures, EsaR/I plasmid from addgene (CHL) and BBa_K299812 from iGEM HQ.
 
<br>
 
<br>
 +
&bull; Streaked out on plates with amp.
 +
<br><br>
 
</p>
 
</p>
 
</td>
 
</td>
 
</tr>
 
</tr>
 +
 +
<tr><td><div style = "border-top: 2px solid turquoise;"></div></td></tr>
  
 
<tr class = "tablenotebook"><td><p class = "paper">
 
<tr class = "tablenotebook"><td><p class = "paper">
−
9th June 2015
+
May 26
 
<br><br>
 
<br><br>
−
<strong>Yi Han</strong>
+
<strong>Adrian</strong>
 
<br><br>
 
<br><br>
−
&bull; All gfp plasmids had a correct sequence<br>
+
&bull; Transformed:<br>
−
&bull; Yun Ting kept glycerol stocok for all, and inoculation of 3mL of gfp3 into 100mL LB+amp for midiprep<br>
+
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;+Kit plate 1 9N Ba_K763002 chl<br>
−
</p>
+
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;+Kit plate 4 13L BBa_E0040 amp
−
</td>
+
<br>
−
</tr>
+
&bull; DNa was received in powder form in plates, and resuspended in 10ul ultrapure H2O respectively. Plates were stored in -20/
−
 
+
−
<tr class = "tablenotebook"><td><p class = "paper">
+
−
10th June 2015
+
 
<br><br>
 
<br><br>
−
<strong>Yi Han & Yun Ting</strong>
+
<strong>Yi Han</strong>
 
<br><br>
 
<br><br>
−
&bull; Midiprep of gfp plasmid  
+
&bull; Plates cracked in incubator as they dried from lack of humidity.
 +
<br>
 +
&bull; Transfer to small incubator with beaker of water for humidity no single colonies for inv plasmid -> streak again.
 
<br>
 
<br>
−
&bull; PCR of gfp with KpnI-gfp and XhoI-gfp primers
+
&bull; Wrong antibiotic for EsaR/I plasmid &rarr; streak out again
 
</p>
 
</p>
 
</td>
 
</td>
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<tr class = "tablenotebook"><td><p class = "paper">
 
<tr class = "tablenotebook"><td><p class = "paper">
−
11th June 2015
+
May 28
 
<br><br>
 
<br><br>
−
<strong>Yun Ting & Duy</strong>
+
<strong>Yi Han</strong>
 
<br><br>
 
<br><br>
−
&bull; Nanodrop of gfp product &rarr; 595.5ng/ul
+
&bull; Inoculate single colony of invasin plasmid carrying bacteria in 3mL LB+amp
 
<br>
 
<br>
−
&bull; RE digest of EsaR vector with KpnI/XHoI<br>
+
&bull; Transformation of 13L repeated with 1ul of DNA.
−
&bull; Gel electrophoreseis at 1000V for 30min<br>
+
−
&bull; Gel extraction: 3.9ng/ul and 6.9ng/ul for Esa fragment and GFP --&gt; low yield<br>
+
 
</p>
 
</p>
 
</td>
 
</td>
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<tr class = "tablenotebook"><td><p class = "paper">
 
<tr class = "tablenotebook"><td><p class = "paper">
−
12th June 2015
+
May 29
 
<br><br>
 
<br><br>
−
<strong>Yun Ting</strong>
+
<strong>Xin Yi</strong>
 
<br><br>
 
<br><br>
−
&bull; Gel extraction using Promega binding solution to melt gel, followed by thermo scientific kit
+
&bull; No colonies grew for 13L on all plates &rarr; adjust incubator, make new media
 +
<br>
 +
&bull; Miniprep of inoculated bacteria for invasin plasmid, incubate sample in 37degC for 1h 20min:
 
<br><br>
 
<br><br>
−
<strong>Adrian</strong>
+
<div>
 +
<center>
 +
<table class = "nbtable">
 +
<tr><td><b>RE of invasin plasmid</b></td>
 +
<td><b>RE control</b></td></tr>
 +
<tr><td>Rsal 1ul</td>
 +
<td>Rsal 0 ul</td></tr>
 +
<tr><td>EcoRI 1 ul</td>
 +
<td>EcoRI 0 ul</td></tr>
 +
<tr><td>INv plasmid 7.5ul</td>
 +
<td>Inv plasmid 7.5 ul</td></tr>
 +
<tr><td>H2O 35.5 ul</td>
 +
<td>H2O 37.5 ul</td></tr>
 +
<tr><td>NEB buffer 4.5 ul</td>
 +
<td>Buffer 4.5 ul</td></tr>
 +
</table></center>
 +
</div>
 +
<br>
 +
<div class = "nbafter">&bull; Repeat transformation of 13L with 1ul</div>
 
<br><br>
 
<br><br>
−
&bull; Gel extraction optimisation
 
−
<br>
 
−
&bull; Hypothesised that the Binding buffer has a problem/DNA does not bind to column
 
−
<br>
 
−
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 
−
1: 2X promega binding buffer volume<br>
 
−
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 
−
2: Increase incubation time for binding to 5min
 
−
<br>
 
−
&bull; Switch binding buffer to that of thermo scientific PCR purification kit<br>
 
−
&bull; Use sodium acetate if available? To facilitate stronger binding to column
 
−
<br>
 
−
<u>Results:</u><br>
 
−
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 1: ~10ng/ul<br>
 
−
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 2: ~9ng/ul<br>
 
−
&bull;Further optimisation-&gt; warm buffer, incubate for 5min<br>
 
−
&bull;Elute in 30/20ul smaller volumes<br>
 
 
</p>
 
</p>
 
</td>
 
</td>
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<tr class = "tablenotebook"><td><p class = "paper">
 
<tr class = "tablenotebook"><td><p class = "paper">
−
13th June 2015
+
May 30
 
<br><br>
 
<br><br>
−
<strong>Yi Han</strong>
+
<strong>Xin Yi & Yi Han</strong>
 
<br><br>
 
<br><br>
−
1: Thermoscientific miniprep columns with 2XThermoscientific binding buffer
+
&bull; Miniprep of EsaR/I plasmid for 4 colonies
 
<br>
 
<br>
−
2: Thermoscientific PCR purification kit 2X buffer
+
&bull; Restriction digest for EsaR with XbaI/BamHI:
−
<br>
+
<br><br>
−
3: Promega kit 2X buffer
+
<div>
−
<br><br><br>
+
<center>
 +
<table class = "nbtable">
 +
<tr><td><b>RE reaction</b></td></tr>
 +
<tr><td>5ul plasmid</td></tr>
 +
<tr><td>5ul buffer</td></tr>
 +
<tr><td>1ul XbaI</td></tr>
 +
<tr><td>1ul BamHI</td></tr>
 +
<tr><td>Add H2O to 50ul</td></tr>
 +
</table></center>
 +
</div>
 +
<br><br>
 
</p>
 
</p>
 
</td>
 
</td>
 
</tr>
 
</tr>
 +
  
 
</table>
 
</table>

Revision as of 16:15, 13 September 2015


Research Notebook

Week 1 (24/5 - 30/5)

May 25

[Yi Han]

• Received 2 bacterial stab cultures, EsaR/I plasmid from addgene (CHL) and BBa_K299812 from iGEM HQ.
• Streaked out on plates with amp.

May 26

Adrian

• Transformed:
      +Kit plate 1 9N Ba_K763002 chl
      +Kit plate 4 13L BBa_E0040 amp
• DNa was received in powder form in plates, and resuspended in 10ul ultrapure H2O respectively. Plates were stored in -20/

Yi Han

• Plates cracked in incubator as they dried from lack of humidity.
• Transfer to small incubator with beaker of water for humidity no single colonies for inv plasmid -> streak again.
• Wrong antibiotic for EsaR/I plasmid → streak out again

May 28

Yi Han

• Inoculate single colony of invasin plasmid carrying bacteria in 3mL LB+amp
• Transformation of 13L repeated with 1ul of DNA.

May 29

Xin Yi

• No colonies grew for 13L on all plates → adjust incubator, make new media
• Miniprep of inoculated bacteria for invasin plasmid, incubate sample in 37degC for 1h 20min:

RE of invasin plasmid RE control
Rsal 1ul Rsal 0 ul
EcoRI 1 ul EcoRI 0 ul
INv plasmid 7.5ul Inv plasmid 7.5 ul
H2O 35.5 ul H2O 37.5 ul
NEB buffer 4.5 ul Buffer 4.5 ul

• Repeat transformation of 13L with 1ul

May 30

Xin Yi & Yi Han

• Miniprep of EsaR/I plasmid for 4 colonies
• Restriction digest for EsaR with XbaI/BamHI:

RE reaction
5ul plasmid
5ul buffer
1ul XbaI
1ul BamHI
Add H2O to 50ul