Biology 475 Molecular Biology Lab Four: Large-Scale Plasmid Midi Prep, DNA Fingerprinting Elizabeth Jacobsmuhlen Spring 2003

Introduction This lab procedure allows for extraction of plasmid DNA that results in a larger and cleaner DNA product than that obtained in lab two. DNA fingerprinting will be performed which will help us to determine if the enzyme cocktails were effective. The DNA product obtained this week will also be used in lab five for DNA sequencing.

	Midi-Prep Methods
Methods: Picture to the right is of me placing my two samples already in50 ml conical tubes into the multi-port vacuum device. One tube for each sample clone included: supernatant containing plasmid DNA and other materials not removed by pelleting, resuspension, and filtering through a cheesecloth, and resin.
Methods: After the conical tubes were removed from the multi-port vacuum device this is what the sample looked like. In each tube is the sample DNA stuck to the resin beads by chemical bonding attractions. These tubes were then cut and had very warm water ran over them to remove the DNA from the resin beads.

Restriction Enzyme Methods

Figure Out	HaeIII	HhaI	HindIII
Recognize…	GG↓CC CC↑GG	G CG ↓C C↑GC G	A↓AGCT T T TCGA ↑A
Bacterial Source	Haemophilu 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^o Celsius	37^o Celsius	37^o Celsius
# Cuts per 5 kb	(1/4)^4 x (5000) bp = 19.53 cuts	(1/4)^4 x (5000) bp = 19.53	(1/4)^6 x (5000) bp = 1.22 cuts

Recipes	DNA	Enzyme(s)	Buffer & Amount	Water
Uncut	5 ul	No enzyme	No buffer	10.0 ul
HaeIII only	5 ul	0.3 ul	Buffer #2 Amount: 1.5 ul	8.2 ul
HhaI only	5 ul	0.3 ul	Buffer #2 Amount: 1.5 ul	8.2 ul
HindIII only	5 ul	0.3 ul	Buffer #2 Amount: 1.5 ul	8.2 ul
HhaI/HindIII	5 ul	0.3 ul HhaI 0.3 ul Hind III	Buffer #2 Amount: 1.5 ul	7.9

	Restriction Methods and Results
Digestion Methods: Isolated plasmid DNA from the large-scale plasmid midi prep was loaded onto an electrophoresis gel. The samples loaded contained one or more of the restriction enzymes, DNA from one of the two samples (#73 or #74), buffer,water, and dye. One lane contained molecular weight standard.
Results: Lane 1: molecular weight standard Lane 2: uncut clone #73 Lane 3: uncut clone #74 Lane 4: Hae III clone #73 Lane 5: Hae III clone #74 Lane 6: Hha I clone #73 Lane 7: Hha I clone #74 Lane 8: Hind III clone #73 Lane 9: Hind III clone #74 Lane 10: Hha I & Hind III clone #73 Lane 11: Hha I & Hind III clone #74

Discussion

This lab resulted in a larger and cleaner DNA product. E. coli was grown in ampicillin media before we received it. These E. coli had plasmids that contained insert-postitive isolates from the mini-prep; the plasmids were then extracted through cell lysis; the DNA extracted; and then the DNA was cleaned through use of resin and vacuum system. Plasmid DNA was purified and isolated using restriction enzymes and the methods described above. In order to isolate the plasmid DNA, template DNA, enzymes (Hae III, Hha I, and Hind III), enzyme buffer (buffer #2), and water are required. This method allows for the extraction of a large and purified DNA product.

Lanes 2 and 3 appear to be uncut as expected. Lane 7, 8, and 9 also appear to be uncut. This could indicate that Hha I was not added. However, it could indicate that for Clone #74 Hha I was not a very effective restriction enzyme and Hind III was not very effective for both Clone #73 and #74. Further experimentation would need to be conducted to determine whether or not Hind III is effective for Clone #73 and #74. Lane 4, 5, and 6 indicates that Hae III was successful for both clone #73 and #74 and Hha I was a successful restriction enzyme for clone #73.

These enzymes are useful in helping to determine if clone #73 and clone #74 were from related bacteria. Hae III was successful for both #73 and #74 and Hha I + Hind III was successful for both #73 and #74, this indicates that the two clones were most likely from related bacteria. However, Hha I + Hind III was more successful for clone #74 than it was for clone #73 this indicates that these two samples may not be from the same type of bacteria.