User:Eric Ma/Notebook/MICB323 Lab Book/2009/01/20

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Lab 2: PCR Gel Electrophoresis, PCR: Ligation, Bacteriophage Growth Curve Analysis

Part 1: Electrophoresis of PCR Products

Gel Ingredients:

  • 100mL 1X TAE
  • 1.5g agarose
  • 10µL SYBRsafe

Lanes:

  • 1: SD 1
  • 2: SD 2
  • 3: SD 3
  • 4: SD 4
  • 5: Mass Ruler
  • 6: ES 1
  • 7: ES 2 - 380 bp product appeared on gel
  • 8: ES 3
  • 9: ES 4 - 1000 bp product appeared on gel
  • 10: ES 5
  • 11: Mass Ruler
  • 12: EM 1: 1X KCl (0.25M)
  • 13: EM 2: 0X KCl
  • 14: EM 3: 0.5X KCl
  • 15: EM 4: 4X KCl

Run gel, 120V for 1hr. Gel Image:

Gel Image.

It seems that there is some effect of KCl on PCR product concentration. Increasing to 4X eliminates the presence of PCR product. Maybe 2X to 3X KCl should be tried to see where cutoff is.

Part 2: Ligation Reaction

  • Followed protocol in lab manual - pages 25 to 26.

Part 3: Bacteriophage Growth Curve

Raw data and calculations are found here:


Calculation of Burst Size:

  • Lower step average: 5x102 pfu/mL
  • Upper step average: 3.5x104 pfu/mL
  • Average burst size = 3.5x104/5.0x102 = 70
  • This is approximately the expected range. (Mrs. Hinze told us average burst size is around 100 for Phage T4 in E. coli).


Calculation of Input MOI:

  • (1.0x108 pfu/ml x 0.1ml) / (1.0x108 cells/ml x 1.0ml) = 0.1 pfu/cell


Calculation of Adsorbed MOI:

  • Number of unadsorbed phage= 51.5pfu x 101ml-1 = 515 pfu/ml
  • Number of infected cells = 7.5 infected cells x 101ml-1 = 75 infected cells/ml (ESPC)
  • [T4]= 1.0x108 pfu/ml x 0.1ml = 1.0x107 pfu / (1.1ml x 104 diliution)= 909.09 pfu/ml
  • Adsorbed moi= (909.09 pfu/ml - 515 pfu/ml) / 75 infected cells/ml = 5.25 pfu/cell (ESPC)

Note to self: Calculations - still need to figure out how to display calculations in wiki format. For now, viewer can download excel file to view formulas used for the calculations.

Summary

  1. Gel electrophoresis went well - PCR products showed up on my own and in the joint samples. Sunny will be using my data for his own analysis.
  2. Ligation reaction proceeded without much fanfare. Need to wait till next lab to see ligation result.
  3. Bacteriophage growth curve produced a beautiful graph. So did Mark's curve. Sunny will be re-plotting the graph to get the nice smooth curve (that doesn't necessarily go through all the points) to help us more accurately determine phage burst size, adsorbed MOI etc.


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