Biology 475

Molecular Biology

Lab Four - Plasmid Midi-Prep

and DNA Fingerprinting

Shanna Briggs

Introduction

The goal of this lab was to isolate and purify DNA from E. coli clones and analyze that DNA using DNA fingerprinting methods. I used clone number 82.

	Midi-Prep Methods
Methods: The bacteria spun down at 10,000 rpm for 10 minutes at 4 C and the supernatant was discarded. The remaining pellet of bacteria cells were allowed to air dry for approximately one minute before resuspension and lysing.
Methods: After lysing the cells the solution was centrifuged again at 12,000 rpm for 20 minutes. The supernatant was transferred to a sterile 50 ml conical tube by filtering through cheese cloth. Following filtration, DNA-binding resin was added to the solution to isolate the plasmids.

Restriction Enzymes Methods

Figure Out	HaeIII	HhaI	HindIII
Recognizes…	GG↓CC CC↑GG	G CG↓C C↑GC G	A ↓AGCT T T TCGA↑A
Bacterial Source	Haemophilus aegyptius	Haemophilus haemolyticus	Haemophilus influenza
Best Buffer	RE_ACT 2	RE_ACT 2	RE_ACT 2
Next Best Buffer	RE_ACT 1, 4, 6 (100%)	RE_ACT 1,3,7 (100%)	RE_ACT 1 (55%)
Units/ul	10 units/ul	10 units/ul	10 units/ul
Cost	$63/ 2500 units	$60/ 1500 units	$23/ 5000 units
Reaction Temp.	37º C	37º C	37º C
# Cuts per 5 kb	(1/4)⁴(5000) =19.53 cuts	(1/4)⁴(5000) =19.53 cuts	(1/4)⁶(5000) =1.22 cuts

Recipes	DNA	Enzyme(s)	Buffer & Amt	Water
Uncut	5 ul	None	None	10.0 ul
HaeIII only	5 ul	0.3 ul	1.5 ul	8.2 ul
HhaI only	5 ul	0.3 ul	1.5 ul	8.2 ul
HindIII only	5ul	0.3 ul	1.5 ul	8.2 ul
HhaI/HindIII	5ul	0.3 ul	1.5 ul	7.9 ul

Restriction Methods and Results

Digestion Methods: The recipes above were mixed and incubated for one hour. Loading dye (blue) was added to each sample to weight down sample and making loading easier. They were loaded on an agarose gel.

Results: Lane 1 (starting at the far left) has the standard. Lane 2 has the uncut plasmid DNA with insert. Lane 3 was cut with Hae III only, lane 4 was cut with Hha I only, lane 5 was cut with Hind III only and lane 6 was cut with Hha I and Hind III.

1 2 3 4 5 6

Discussion

The overall goal of this lab was to perform DNA Fingerprinting, a technique based on Restriction Fragment Length Polymorphism (RFLP) analysis on the cloned insert. To do so, we had to isolate and purify a large amount of plasmid DNA. This was accomplished by growing the E.coli in media with ampicillin, spinning down the bacteria, lysing the cells and pelleting the cellular debris and genomic DNA. The plasmids were isolated from the supernatant with DNA binding resin. The plasmids were then cut with restriction enzymes. Restriction enzymes recognize a specific DNA sequence and cut the DNA at that sequence. To separate the fragments resulting from the various restriction enzymes (see table above) by size, we ran them through gel electrophoresis.

Bands on the gel show that the isolation of DNA was successful because the DNA is present. This gel also demonstrates the effectiveness of the enzymes Hae III and Hha I. As expected from the calculations above, these enzymes produced fragments of approximately the same size and thus have bands at similar locations on the gel. Also as predicted, Hind III makes fewer cuts and results in larger fragments which travel slower on the gel. The gel also demonstrates that uncut DNA, which is tightly coiled, travels farther through the gel than does cut DNA. The bright, highly concentrated bands in each column indicate that all three enzymes cut well.

This gel alone reveals very little about the specific 16S insert. However, there are some distinct similarities in banding patterns with gels from other students. This indicates that the bacteria from which the insert was isolated are closely related. Based on these results we expect that the sequences determined next week will be very similar as well and that the BLAST hits will overlap.