Biology 475

Biology 475

Molecular Biology

Lab Four – Plasmid Midi-Prep

and DNA Fingerprinting

Kelly L. Shipley

Introduction

In this lab we are going to isolate a large amount of the plasmid DNA, which contains our target 16s rRNA inserts. Based on the DNA Probe Hybridization tests and Restriction Digests we are using sample #52 and #53. In doing the three basic steps of large scale plasmid isolation we will isolate the plasmid for future DNA sequencing. After isolating the DNA we will use enzymes to cut our DNA at specific sites and run them in electrophoresis gels to determine the size and variety of inserts.

Midi-Prep Methods

Methods: After centrifuging our cells into a pellet, we resuspend them using Resuspension Buffer and pipetting up and down gently using a disposable pipette.

Methods: The multi-port vacuum device created by Danny is used to pull our target plasmid from the supernatant of our solution down through the tube using the vacuum. DNA-binding Resin is added to the solution, the vacuum is turned on and all plasmid DNA bound by the resin is now too large to go through the filter on the bottom of the tube. At the end of this process all of our target plasmid DNA is in the tube above the filter.

Restriction Enzymes Methods

Figure Out:	HaeIII	Hhal	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	# 2 (100%)	# 2 (100%)	# 2 (100%)
Next Best Buffer	#1, #4, #6 (all 100%)	#1, #3, #7 (all 100%)	#1 (55%)
Units / ul	10 units / ul	10 units / ul	10 units / ul
Cost	$63.00 / 2500 units	$60.00 / 1500 units	$23.00 / 5000 units
Reaction Temp.	37° C	37° C	37° C
# Cuts per 5 kb	(1/4)^{4 (x)}5000 bp = 19.53 cuts	(1/4)^{4 (x)}5000 bp = 19.53 cuts	(1/4)^{6 (x)}5000 bp = 1.22 cuts

Restriction Enzyme Cocktails

Recipes:

DNA

Enzyme(s)

Buffer & Amt.

Water

Uncut

5 ul

No enzyme

No buffer

10.0ul

HaeIII only

5 ul

0.3 ul

Buffer #2

Amt. 1.5 ul

8.2 ul

Hhal only

5 ul

0.3 ul

Buffer #2

Amt. 1.5 ul

8.2 ul

HindIII only

5 ul

0.3 ul

Buffer #2

Amt. 1.5 ul

8.2 ul

Hhal/HindIII

5 ul

0.3 ul

Buffer #2

Amt. 1.5 ul

7.9 ul

Restriction Methods and Results

Digestion Methods: Restriction Enzyme cocktails are made using the above necessary ingredients; specific restriction enzyme (we used HaeIII, HhaI and HindIII), water, buffer (React #2 ), and our sample DNA (#52).

Results: Sample #52 was digested and run through gel electrophoresis.

Lane M: DNA marker lane

Lane 1: Uncut DNA, some small fragments seen.

Lane 2: DNA cut with HaeIII, shows a large band of DNA much smaller than original DNA.

Lane 3: DNA cut with HhaI shows “smeared” band indicating similarity in size between different pieces of DNA.

Lane 4: DNA cut with HindIII was only supposed to cut the 5000 bp sequence once on average.

Lane 5: DNA cut with HindIII and HhaI. The band is about the same size as the pieces cut by HhaI.

Discussion

In order to sequence our target DNA in the future, we needed to isolate the sequence and rapidly amplify the amount which we had. To do this, we first started our procedure today with the Midi Prep which is designed to give us DNA product which is compatible with our sequencing machinery. Plasmid isolation at this large scale level still encompasses the same basic steps as our previous mini prep; lysing, separating, and purifying.

Using restriction enzymes HaeIII, HhaI, and HindIII we cut Sample #52. We did single digest using each of them and one double digest using both HhaI and HindIII together. The formula for the cocktail used for mixing the restriction enzymes with our DNA samples is located in the Results section above. After letting the restriction digests cut our sample DNA we proceeded to loading the agarose gel. In Lane 1 we had DNA marker, Lane 2 Uncut DNA, Lane 3 HaeIII, Lane 4 HhaI, Lane 5 HindIII and Lane 6 HhaI and HindIII together. After the gel had run, we had a map of where and how the DNA had been cut by the restriction enzymes; this is called a Restriction Fragment Length Polymorphism.

Unfortunately, I did not use my Sample #53 to create an RFLP also, so I can not compare my results from Sample #52 to another sample, but can say in looking at other students RFLPs, that there were some identical to Sample #52, and some which were extremely different. Even without going any further with this research, we can conclude from the differences that there is definite diversity within the Yellowstone microbial mat bacterial population.

From Sample #52 and its’ RFLP I can make some definite observations. In Lane 1, the uncut DNA did not travel as far as I believed it should have. If the DNA had been better prepared, i.e. more super coiled in its’ configuration, I believe it would have traveled farther. Also there are evident smaller bands in the uncut DNA lane, telling us that the DNA was damaged at some point during preparation and smaller pieces were created. Lane 2 was cut with HaeIII, which we statistically figured would create base pair sequences approximately 256 bp long. This calculation was also the same for DNA cut with HhaI in Lane 3. We can see from our RFLP that there are bands corresponding to a much smaller weight (i.e. traveled farther) on the gel in Lanes 2 and 3. The smeared appearance of these lanes gives the idea that the cuts were not exactly at 256 bp each time, but approximately, which is expected. Lane 4 shows DNA cut with HindIII. HindIII was only calculated to cut approximately once per 5000 bp. From this calculation, I expected to see a band at a smaller weight than the uncut DNA and another band corresponding to a very small amount of base pairs. On the RFLP we see no band corresponding to the smaller pieces, and a very distinct band at a weight seemingly more than the uncut DNA. This is backwards from the expected results, indicating a problem in preparation or execution of the procedure. Lastly, in Lane 5 we have the double digest. Here we see clearly a band at the same weight as Lanes 2 and 3 giving the impression that the sample was cut with HindIII and then with the HhaI, giving many more distinct pieces approximately 256 bp in length.

In creating an RFLP for a target sequence, we can use many different restriction enzymes separately, then together to see what types of pieces we come up with. In doing this we can start to map out the sequence of the DNA, at least start to understand which restriction sites are there, which genes they may follow or precede. By cutting with certain restriction enzymes and then testing our samples for the presence of functional lac Z or AntR genes, we can determine where those genes may be within our target DNA.