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

Line 13: Line 13:
 
<li>Follow the standard protocol of Thermo Scientific miniprep kit <font color="red">(is it? I followed Ihab's protocol here: https://docs.google.com/document/d/1QHBHcN8RY0c-oEY8X5kAqPrKYODTBm7K8eU_lY_yw-o/edit, we should compare them)</font></li>
 
<li>Follow the standard protocol of Thermo Scientific miniprep kit <font color="red">(is it? I followed Ihab's protocol here: https://docs.google.com/document/d/1QHBHcN8RY0c-oEY8X5kAqPrKYODTBm7K8eU_lY_yw-o/edit, we should compare them)</font></li>
 
<li>Measure concentration with Nanodrop</li>
 
<li>Measure concentration with Nanodrop</li>
−
 
 
</ol>
 
</ol>
  
Line 24: Line 23:
 
<li>tube HO pl. 4 = 261.7 ng/uL</li>
 
<li>tube HO pl. 4 = 261.7 ng/uL</li>
 
</ul>
 
</ul>
 +
 +
<br><br>
 +
<b>Jul. 15th</b>
 +
 +
<h3>Goal</h3>
 +
Test chromosomal integration in WT yeast SK1 with the integrative plasmid HO-Poly-KanMX4-HO.
 +
 +
<h3>Procedure</h3>
 +
<font color="red">JB can you fill this? <3 Thanks!</font>
 +
<ol>
 +
<li>Step 1</li>
 +
<li>Step 2</li>
 +
</ol>
 +
 +
<h3>Results</h3>
 +
We had many transformants, with the resistance marker:
 +
<br><font color="red">[add photos]
 +
<br>we must not forget to mention the controls.</font>
 +
  
 
<br><br>
 
<br><br>

Revision as of 10:04, 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. 15th

Goal

Test chromosomal integration in WT yeast SK1 with the integrative plasmid HO-Poly-KanMX4-HO.

Procedure

JB can you fill this? <3 Thanks!
  1. Step 1
  2. Step 2

Results

We had many transformants, with the resistance marker:
[add photos]
we must not forget to mention the controls.

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

Results

After gel migration, we got this: [image]
The PCR worked.

We then made a PCR purification (should I describe full protocol?) and measured the final concentration of gBlocks with a Nanodrop:
  • Final concentration gBlock vA-2 tube a = 126.1 ng/uL
  • Final concentration gBlock vA-2 tube b = 96.6 ng/uL
  • Final concentration gBlock vA-3 tube a = 128.7 ng/uL
  • Final concentration gBlock vA-3 tube b = 75.8 ng/uL
  • Final concentration gBlock vA-4 tube a = 201.8 ng/uL
  • Final concentration gBlock vA-4 tube b = 108.3 ng/uL