Difference between revisions of "Team:Paris Bettencourt/Notebook/VitaminA"

Line 23: Line 23:
 
<li>tube HO pl. 3 = 366.4 ng/uL</li>
 
<li>tube HO pl. 3 = 366.4 ng/uL</li>
 
<li>tube HO pl. 4 = 261.7 ng/uL</li>
 
<li>tube HO pl. 4 = 261.7 ng/uL</li>
 +
</ul>
  
 
<br><br>
 
<br><br>
−
<b>Jul. 24th</b>
+
<b>Jul. 22nd</b>
 +
 
 +
<h3>Goal</h3>
 +
Amplify gBlocks vA-2, vA-3, and vA-4, which form the last parts of the polycistron.
 +
 
 +
<h3>Procedure</h3>
 +
<b>Resuspend gBlocks and primers:</b>
 
<ul>
 
<ul>
−
<li>Made the project, it is awesome!</li>
+
<li>Resuspended gBlocks vA-2, vA-3 and vA-4 in 100 uL water, to reach a final concentration of 10 ng/uL.</li>
 +
<li>Made aliquots of these gBlocks at concentration 1 ng/uL.</li>
 +
<li>Resuspended primers o15.056, o15.076, o15.058, o15.059, o15.060, o15.061 in water to reach a final concentration of 100 uM for each.</li>
 +
<li>Made aliquots of each primer at concentration 10 uM.</li>
 
</ul>
 
</ul>
 +
 +
<br><b>PCR:</b>
 +
Made 2 tubes of PCR for each gBlocks, containing:
 +
<ul>
 +
<li>1 uL of gBlock (1 ng)</li>
 +
<li>2 uL forward primer</li>
 +
<li>2 uL reverse primer</li>
 +
<li>50 uL Master Mix 2X</li>
 +
<li>45 uL water</li>
 +
</ul>
 +
 +
<br><b>Settings PCR:</b>
 +
<ul>
 +
<li>30s at 98°C</li>
 +
<li>35 times:
 +
<ul>
 +
<li>10s at 98°C</li>
 +
<li>30s at 50°C</li>
 +
<li>1m at 72°C</li>
 +
</ul></li>
 +
<li>10m at 72°C</li>
 +
<li>the tubes were then kept at 10°C</li>
 +
</ul>
 +
 +
<br><br>
 +
<b>Jul. 22nd</b>
 +
 +
<h3>Goal</h3>
 +
Check if PCR of gBlocks vA-2, vA-3 and vA-4 worked.

Revision as of 08:47, 24 July 2015

Jul. 14th

Goal

Extract the integrative plasmid HO-Poly-KanMX4-HO from the E.Coli provided by AddGene (Accession number #51662).

Procedure

  1. Liquid culture overnight in LB + Ampicillin.
  2. Made a glycerol stock and stored it in the -20 freezer (g15.35)
  3. Centrifuge the tube for 1 minute with 11000 rpm.
  4. Follow the standard protocol of Thermo Scientific miniprep kit (is it? I followed Ihab's protocol here: https://docs.google.com/document/d/1QHBHcN8RY0c-oEY8X5kAqPrKYODTBm7K8eU_lY_yw-o/edit, we should compare them)
  5. Measure concentration with Nanodrop

Results

Final DNA concentrations of the 4 tubes of miniprep, measured with Nanodrop:
  • tube HO pl. 1 = 431.1 ng/uL
  • tube HO pl. 2 = 313.6 ng/uL
  • tube HO pl. 3 = 366.4 ng/uL
  • tube HO pl. 4 = 261.7 ng/uL


Jul. 22nd

Goal

Amplify gBlocks vA-2, vA-3, and vA-4, which form the last parts of the polycistron.

Procedure

Resuspend gBlocks and primers:
  • Resuspended gBlocks vA-2, vA-3 and vA-4 in 100 uL water, to reach a final concentration of 10 ng/uL.
  • Made aliquots of these gBlocks at concentration 1 ng/uL.
  • Resuspended primers o15.056, o15.076, o15.058, o15.059, o15.060, o15.061 in water to reach a final concentration of 100 uM for each.
  • Made aliquots of each primer at concentration 10 uM.

PCR: Made 2 tubes of PCR for each gBlocks, containing:
  • 1 uL of gBlock (1 ng)
  • 2 uL forward primer
  • 2 uL reverse primer
  • 50 uL Master Mix 2X
  • 45 uL water

Settings PCR:
  • 30s at 98°C
  • 35 times:
    • 10s at 98°C
    • 30s at 50°C
    • 1m at 72°C
  • 10m at 72°C
  • the tubes were then kept at 10°C


Jul. 22nd

Goal

Check if PCR of gBlocks vA-2, vA-3 and vA-4 worked.