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

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*Note: Sunny will do the p421 and CaCl<sub>2</sub>
*Note: Sunny will do the p421 and CaCl<sub>2</sub>
*Protocol followed as outlined in Lab Manual pages 37-38
*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
<br>
#Plates into 4ºC fridge at 11AM on 28 Jan 2009


==Summary of Plates==
==Summary of Plates==

Revision as of 12:18, 28 January 2009

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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

  1. Plates into 37ºC incubator at: 4PM on 27 Jan 2009
  2. Plates out of 37ºC incubator at: 10AM on 28 Jan 2009


  1. Plates into 4ºC fridge at 11AM on 28 Jan 2009

Summary of Plates

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 Part 2

Expt. Condition Plate Dilution Plated (10x) Final Plated Volume (mL) Final Plated Dilution (10x) # Blue Colonies # White Colonies Total # of Colonies Total Cell Titre (cfu/mL) Notes
Pre-Incubation Original Cells LB -5 0.1 -6 0 247 247 2.47E+08
Pre-Incubation Original Cells LB -6 0.1 -7 NC NC NC NC
Transformed LB-AMP + X-gal 0 0.1 -1 4 TNTC TNTC TNTC
Transformed LB-AMP + X-gal -1 0.1 -2 NC TNTC TNTC TNTC
Transformed LB-AMP + X-gal -2 0.1 -3 0 261 261 2.61E+05
Transformed LB -3 0.1 -4 TNTC TNTC TNTC TNTC
Transformed LB -4 0.1 -5 0 102 102 1.02E+07
Transformed LB -5 0.1 -6 NC NC NC NC
Untransformed LB-AMP + X-gal 0 0.1 -1 0 0 0 0.00E+00
Untransformed LB -3 0.1 -4 NC TNTC TNTC TNTC
Untransformed LB -4 0.1 -5 0 134 134 1.34E+07
Untransformed LB -5 0.1 -6 NC NC NC NC
Post-Incubation Original Cells LB -6 0.1 -7 0 16 16 1.60E+08 ESPC
Post-Incubation Original Cells LB -7 0.1 -8 NC NC NC NC

See this Excel spreadsheet to see how calculations were done.

Summary

  1. Plating went well and smoothly.
  2. Electroporation is surprisingly easy! Just push that button. "That was easy!"


I have a few questions:

  1. What is the purpose of the pre-incubation plating and post-incubation plating of the original culture? Is it some control of sorts?
  2. The purpose of plating on LB alone - to get a feel for the total number of cells present?
  3. 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

  1. 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.
  2. Selected pure white colonies from p421 transformants. Pick them from the "Part 2(a)" plate on Monday.



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