Biology 475

Molecular Biology

Lab Four – Plasmid Midi-Prep

and DNA Fingerprinting

Rogan Rattray

Introduction

We will be using restriction enzymes to digest the vector plasmids which have the cDNA 16S gene insert. We will then run these digested samples through agarose gel by electrophoresis to check for general differences in the clonal populations’ inserted sequence.

	Mini-Prep Methods
Methods: The incubated colonies were transferred from the flasks they were growing in to round bottom centrifuge tubes (right) where the cells were spun down out of the liquid media. The media supernatant was then discarded and the pelleted cells were resuspended in a buffer solution to which lysozyme was added to gently break open the cells.
Methods: Large cell debris was separated from the plasmid DNA by centrifugation. Smaller debris and proteins were separated from the DNA by columnar purification. This method utilized a DNA binding resin as the column packing to which the plasmids bound to as the solutions were pulled through the column by vacuum filtration. The plasmids were then washed off of the resin using extremely pure hot water.

Restriction Enzyme Methods

	Hae III	Hha I	Hind III
Recognizes Seq.	5' GGCC 3'	5' GCGC 3'	5' AAGCTT 3'
Bacterial Source	Haemophilus aegyptius	Haemophilus haemolyticus	Haemophilus influenzae
Best Buffer	2	2	2
2nd Best Buffer	1	1	1
Units/uL	10	10	10
Cost	$63 for 2,500 units	$60 for 1,500 units	$23 for 5,000 units
Reaction Temp	37 degrees C	37 degrees C	37 degrees C
# Cuts per 5kb	19.5	19.5	1.22

Recipe	DNA	Enzyme	Buffer & Amt	Water
Uncut	5ul	0	1.5ul	8.5ul
Hae III only	5ul	.3ul	1.5ul	8.2ul
Hha I only	5.0ul	0.3ul	1.5ul	8.2ul
Hind III only	5.0ul	0.3ul	1.5ul	8.2ul
Hha I/Hind III	5.0ul	.3ul Hh/.3ul Hi	3.0ul	6.4ul

	Restriction Methods and Results
Digestion Methods: The purified plasmids were added to the different combinations of restriction enzyme cocktails that were prepared (see table above). All of the digests were allowed to incubate in a 37 degrees C water bath for about an hour (right).
Results: The photo to the right represents the gel that was run with the different samples and enzyme combinations. From left to right, the first lane was loaded with molecular weight standards. Lanes 2 and 3 had DNA sample but no enzymes of any kind. Lanes 4 and 5 had DNA with only Hind III. Lanes 6 and 7 had DNA with only Hha I. Lanes 8 and 9 had DNA with only Hae III. Lanes 10 and 11 had DNA and both Hha I and Hind III. For each pair of lanes (except lane 1) sample 49 was in the left lane while sample 50 was in the right.

Discussion

In the first two lanes of the gel that contained DNA sample (2 & 3) we would not expect to see any more bands than one since the DNA was not exposed to any restriction enzyme. The bands that appear in lane 2 (sample 49) and lane 3 (sample 50) are probably a result of not being gentle enough while processing the DNA. Though interesting band patterns were present in lanes 4 and 5, lanes 6 through 11 show the samples running to the end of the gel. This result could be due to the enzyme cutting the plasmids into such small fragments of similar size that no separation or resolution could be gained. The choice of using sample 50 for this procedure was probably a mistake since the results of both the Eco R1 restriction digest and gel and the dot-blot macro array hybridization of this sample showed it to be negative for cDNA insertion of the 16S gene into its plasmid. This discrepancy may be the reason for the ambiguous results of sample 49 having a nice banding pattern in lane 4 and not much banding in lane 5.