Copyright 2003

Introduction

The goal of this lab was to isolate and purify DNA from E. coli and do DNA fingerprinting using several restriction enzymes to see how they cut compared to Eco R1. this is a picture of me lysing the E.coli cells to purify the vectors.

	Midi-Prep Methods
Methods: Before we can cut the DNA with restriction enzymes, we must first extract it from the cells. By adding a lysis solution to a concentrated solution of cells, we can rupture the cell and obtain the plasmids from within.
Methods: After obtaining the DNA from the cells, we washed it through a vacuum filter that had DNA binding resign in order to get rid of anything that was not DNA. This ensures ultra pure DNA samples for our restriction digests.

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	React 2	React 2	React 2
Next Best Buffer	React 1, 4, 6	React 1,3,7	React 1
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	About 19	About 19	About 1

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: We used 3 different restriction enzymes to cut our DNA samples with. The table above gives the recipe for what was used in each case. We also ran a lane of uncut gels.

Results: This is a picture of the results from gel electrophoresis of my RFLP. Lane 1 is at the bottom 11 at the top. The bottom 5 lanes are for sample #1 and its different restriction enzymes, and the top 5 lanes are for sample # 5 and its different restriction digests. In depth discussion of results are below.

Discussion

In this lab we used techniques similar to ones that we have used before to run gels and do DNA fingerprinting. What distinguished this lab from other is the type of restriction enzymes we used to cut the DNA, and even using a combination of enzymes that would give a very distinct pattern. In lane 1 of my sample (the very bottom of the picture) was my uncut DNA from sample #1. since there are several bands that are present, it tells me that my DNA was damaged during the preparation. Lane 2 has 2 visible bands which indicates 1 cut by Hae III. Lane 3 has 3 bands, indicating 2 cuts by Hha I. lane 4 and 5 are a little smeared, and hard to tell what cuts were made, if any. The top 5 lanes were for sample # 5 and are all smeared and about the same length. This tells me that there was no cutting that went on, and my sample was in good shape. Overall the procedures went smoothly however I do need to be more gentle with the DNA next time.