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:
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: 5x10^{2} pfu/mL
 Upper step average: 3.5x10^{4} pfu/mL
 Average burst size = 3.5x10^{4}/5.0x10^{2} = 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:
 (1.0x10^{8} pfu/ml x 0.1ml) / (1.0x10^{8} cells/ml x 1.0ml) = 0.1 pfu/cell
Calculation of Adsorbed MOI:
 Number of unadsorbed phage= 51.5pfu x 10^{1}ml^{1} = 515 pfu/ml
 Number of infected cells = 7.5 infected cells x 10^{1}ml^{1} = 75 infected cells/ml (ESPC)
 [T4]= 1.0x10^{8} pfu/ml x 0.1ml = 1.0x10^{7} pfu / (1.1ml x 10^{4} 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
 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.
 Ligation reaction proceeded without much fanfare. Need to wait till next lab to see ligation result.
 Bacteriophage growth curve produced a beautiful graph. So did Mark's curve. Sunny will be replotting 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.
