Lab Transformation Assignment

Each student is responsible for answering Questions 1-4.

1) Determine the Transformation Efficiency (colonies/µg DNA) for the pDrive plasmid (white colonies on +AMP/-IPTG and Blue colonies on +AMP/+IPTG plates.

a. Determine total mass (µg) of pDrive used in transformation.

b. Fraction/Ratio of cell suspension added to the AMP plate

c. Mass of pDrive on the plate.

d. Count the number of colonies

2) Speculate the results you would observe if you inserted a piece of DNA into the Multiple Cloning Site of the pDrive plasmid, then transformed bacteria as we did in this lab.

3) Suppose your used 9 different concentrations of pDrive plasmid to transform bacteria (same protocol as used above) and obtained the data shown below. Based on this data, answer the following questions.

pDrive concentration        Colonies observed
0.00001 ug/ul                      4
0.00005 ug/ul                      12
0.0001 ug/ul                        32
0.0005 ug/ul                        125
0.001 ug/ul                          442
0.005 ug/ul                          542
0.01 ug/ul                            507
0.05 ug/ul                            475
0.1 ug/ul                              516

a. Plot a graph that shows DNA mass vs Transformation Efficiency.

i. Figure legend for your graph that should have 1: Title (what does the graph show); 2) How as data collected (methodology); and 3) How was data analyzed (Error, Calculations, Significance, Sample size, 5).

ii. Clearly describe the trend (ie. Result) you see in the graph:

4) You have cloned the human Insulin (Ins) gene into the pDrive plasmid, and transformed bacteria using (0.45 ug) of this pDrive/Ins (as per the protocol described in this lab). Using the information below, calculate the number of molecules of Insulin that have been cloned into your entire culture after 5 hours of growth after incubation.

a. The transformation efficiency of this reaction is 104 colonies/ µg intact pDrive/Ins plasmid.
b. pDrive plasmid has a copy number of 100 molecules per transformed cell.
c. Following the heat shock the entire 300 µl of cell suspension was used to inculcate a 20 ml of fresh LB broth.
d. Transformed cells enter log phase 60 min after inoculation and begin to replicate on average every 30 min.