Difference between revisions of "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. 15th

Goal

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

Procedure

We followed the method described in "Hight-efficiency Yeast transformation using liAc/SS carrier DNA/PEG method"

1. 1 Inoculation of a single colony of the SK1 yeast strain in liquid YPD overnight on a rotatory shaker at 130 r.p.m and 30°C.
2. 2 After 16 hours the titer of the cell culture was determined. The OD 600nm of a 1/100 dillution (10uL in 1mL) was measured and the cell concentration determined using the formula ( 1*10^6 cells.mL^-1 will give an OD600nm of 0.1)
3. 3 2.5*10^8 cells were added to 50 mL of pre-warmed YPD and incubated for 4 and half hours at 30°C and 130 rpm
4. 4 1.0mL of salmon sperm carrier DNA was denaturated in a heat block at 99°C during 5 min and chill rapidly in ice.
5. 5 the cells where harvested by centrifugation at 3000g for 5 min and resuspended in 25 mL of water and centrifuged again 5min at 3000g. The washing process was repeated again then the cells were resuspended in 1.0 mL of sterile water.
6. 6 the suspension was transfered into a 1.5mL eppendorf tube and centrifuged for 30s at 13,000g and the suppernatent discarded
7. 7 the cells were resuspended in 0.5mL of sterile water. 100uL of

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