Team:Paris Bettencourt/Notebook/VitaminA

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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

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