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

From OpenWetWare
Jump to navigationJump to search
Line 39: Line 39:
| align="center" style="background:#f0f0f0;"|'''Expt. Condition'''
| align="center" style="background:#f0f0f0;"|'''Expt. Condition'''
| align="center" style="background:#f0f0f0;"|'''Plate'''
| align="center" style="background:#f0f0f0;"|'''Plate'''
| align="center" style="background:#f0f0f0;"|'''Dilution Plated (10x)'''
| align="center" style="background:#f0f0f0;"|'''Dilution Plated (10<sup>x</sup>)'''
| align="center" style="background:#f0f0f0;"|'''Final Plated Volume (mL)'''
| align="center" style="background:#f0f0f0;"|'''Final Plated Volume (mL)'''
| align="center" style="background:#f0f0f0;"|'''Final Plated Dilution (10x)'''
| align="center" style="background:#f0f0f0;"|'''Final Plated Dilution (10<sup>x</sup>)'''
| align="center" style="background:#f0f0f0;"|'''# Blue Colonies'''
| align="center" style="background:#f0f0f0;"|'''# Blue Colonies'''
| align="center" style="background:#f0f0f0;"|'''# White Colonies'''
| align="center" style="background:#f0f0f0;"|'''# White Colonies'''

Revision as of 11:50, 28 January 2009

Project name <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

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

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)
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
Post-Incubation Original Cells LB -7 0.1 -8 NC NC NC NC

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?



Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Eric_Ma/Notebook/MICB323_Lab_Book/2009/01/27&oldid=280556"