User:Eric Ma/Notebook/MICB323 Lab Book/2009/01/27

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Lab 3: Plasmid Transformation of Competent Cells and Isolation of Transformants Part 1: Transformation using ligation reaction and one shot INVaF' CaCl2 prepared competent cells Protocol followed just as outlined in the Lab Manual pg 33 Part 2: Transformation via electroporation using supplied p421 plasmid Note: Sunny will do the p421 and CaCl2 Protocol followed as outlined in Lab Manual pages 37-38 Notes Plates into 37ºC incubator at: 4PM on 27 Jan 2009 Plates out of 37ºC incubator at: 10AM on 28 Jan 2009 Plates into 4ºC fridge at 11AM on 28 Jan 2009 Summary of Plates Dummy Demo Section Samples Average Absorbance (A450) 100X Average Absorbance (A450) Calculated [TNFa] (pg/mL) Normalized [TNFa] (pg/mL) 0 hr PBS control 0.086 8.58 -342.36 0.00 Day 1 +PBS control 0.109 10.85 -230.30 0.00 30 min +LPS 0.097 9.73 -285.71 0.00 60 min +LPS 0.102 10.20 -262.32 0.00 90 min +LPS 0.105 10.53 -246.31 0.00 6 hr +LPS 0.404 40.40 1225.37 1225.37 8 hr +LPS 0.131 13.08 -120.69 0.00 Day 1 +LPS 0.227 22.65 1754.93 1754.93 90 min + IFNg 0.081 8.10 -365.76 0.00 Day 1 +IFNg 0.103 10.33 -256.16 0.00 30 min +LPS +IFNg 0.086 8.55 -343.60 0.00 60 min +LPS +IFNg 0.093 9.33 -305.42 0.00 90 min +LPS +IFNg 0.099 9.93 -275.86 0.00 6 hr +LPS +IFNg 0.207 20.73 256.16 256.16 8 hr +LPS +IFNg 0.178 17.83 113.30 113.30 Day 1 +LPS +IFNg 0.263 26.30 2653.94 2653.94 Plates from Part 1 These are CaCl2 transformed cells using INVaF' competent cells and ligation reaction plasmid. 3 plates: Plated Volume (µL) Final Plated Dilution # Blue Colonies # White Colonies Total # of Colonies Total Cell Titre (cfu/mL) Fraction of cells with transformed plasmid Fraction of cells with untransformed plasmid 200 0.2 31 61 92 460 0.66 0.34 100 0.1 NC NC NC NC NC NC 50 0.05 NC NC NC NC NC NC Plates from Parts 2 and 3 These plates come from electroporation using the p421 plasmid. Experimenter Expt. Condition Procedure Plate Dilution Plated (10x) FPV (mL) FPD (10x) # Blue Colonies # White Colonies Total # of Colonies Total Cell Concentration (cfu/mL) Notes Eric Pre-Incubation Original Cells None LB -5 0.1 -6 0 247 247 2.47E+08 Eric Pre-Incubation Original Cells None LB -6 0.1 -7 NC NC N/C N/C Eric Transformed EP LB-AMP + X-gal 0 0.1 -1 4 TNTC N/C N/C Eric Transformed EP LB-AMP + X-gal -1 0.1 -2 NC TNTC N/C N/C Eric Transformed EP LB-AMP + X-gal -2 0.1 -3 0 261 261 2.61E+05 Eric Transformed EP LB -3 0.1 -4 TNTC TNTC N/C N/C Eric Transformed EP LB -4 0.1 -5 0 102 102 1.02E+07 Eric Transformed EP LB -5 0.1 -6 NC NC N/C N/C Eric Untransformed EP LB-AMP + X-gal 0 0.1 -1 0 0 0 0.00E+00 Eric Untransformed EP LB -3 0.1 -4 NC TNTC N/C N/C Eric Untransformed EP LB -4 0.1 -5 0 134 134 1.34E+07 Eric Untransformed EP LB -5 0.1 -6 NC NC NC NC Eric Post-Incubation Original Cells None LB -6 0.1 -7 0 16 16 1.60E+08 ESPC Eric Post-Incubation Original Cells None LB -7 0.1 -8 NC NC N/C N/C Sunny Pre-Incubation Original Cells None LB -5 0.1 -6 0 63 63 6.30E+07 Sunny Pre-Incubation Original Cells None LB -6 0.1 -7 0 4 4 4.00E+07 ESPC Sunny Transformed HS LB-AMP + X-gal 0 0.1 -1 TNTC TNTC N/C N/C Sunny Transformed HS LB-AMP + X-gal -1 0.1 -2 4 30 34 3.40E+03 Sunny Transformed HS LB-AMP + X-gal -2 0.1 -3 0 6 6 6.00E+03 ESPC Sunny Transformed HS LB -4 0.1 -5 0 280 280 2.80E+07 Sunny Transformed HS LB -5 0.1 -6 0 39 39 3.90E+07 Sunny Transformed HS LB -6 0.1 -7 0 3 3 3.00E+07 ESPC Sunny Untransformed HS LB-AMP + X-gal 0 0.1 -1 0 0 0 0.00E+00 Sunny Untransformed HS LB -4 0.1 -5 TNTC TNTC N/C N/C Sunny Untransformed HS LB -5 0.1 -6 0 38 38 3.80E+07 Sunny Post-Incubation Original Cells None LB -5 0.1 -6 0 33 33 3.30E+07 Sunny Post-Incubation Original Cells None LB -6 0.1 -7 0 2 2 2.00E+07 ESPC See this Excel spreadsheet to see how calculations were done. Summary Plating went well and smoothly. Electroporation is surprisingly easy! Just push that button. "That was easy!" I have a few questions: What is the purpose of the pre-incubation plating and post-incubation plating of the original culture? Is it some control of sorts? The purpose of plating on LB alone - to get a feel for the total number of cells present? We can then take total number of transformants (i.e. from the LB-AMP + X-gal) divided by total number of cells used - get a feel for efficiency of transformation? Plates selected Selected pure white and pure blue colonies from ligation reaction transformants. Prospective ones are labeled on the "Part 1" plate with "W" and "B" respectively. Selected pure white colonies from p421 transformants. Pick them from the "Part 2(a)" plate on Monday.