Mentor:

Dr. George
Clokey
Simon Kara Serenity
Schmidt Kamps Mutchler

RESOURCE PARTITIONING: ♦Tail tissue samples of P. maniculatus and P. leucopus Figure 1: DNA Analysis.
♦A strategy developed to reduce competition for were obtained from the Peromyscus Genetic Stock
PCR amplified DNA from
resources between two species with similar Center.
ecologic niches that occupy the same area. ♦Approximately 3 mm of tissue were clipped from each P. leucopus and P.
♦Previous studies suggest that resource partitioning tail. maniculatus tissue were run as
described. Lanes 1 & 6,
does occur between Peromyscus maniculatus
DNA PREP: Loading dye with Xylene
♦Samples were ground on ice in 0.5 ml of normal Cyanol and Bromophenol
(deer mouse), and Peromyscus leucopus
saline using a micro-pestle in a 1.5 ml microfuge blue; lanes 2 & 5, pGEM
(white-footed mouse).
tube
•Based on tail morphology which can be DNA markers (Promega,
♦0.5 ml 10% Chelex was added
problematic due to the two species being Madison, WI); lane 3, P.
♦Samples were incubated in a boiling water bath for
almost identical (see images below). leucopus PCR amplified DNA
10 minutes
Overlapping Range: mice show resource partitioning and lane 4, P. maniculatus
♦Tissue debris and Chelex were pelleted by a 30
with P. maniculatus occupying the trees and P. PCR amplified DNA.
leucopus occupying the ground second spin down in an Eppendorf microcentrifuge
at 14,000 rpm
[Mg++ ] Well Set 1 Well Set 2
♦The supernatant (ca 0.5 ml) was saved and the
Range of Table 1:
P. maniculatus:
Range of pellet discarded. [Mg++] for the 3.5 mm Lane 1 Lane 1
P. leucopus:
mouse PCR reactions are
occupies both
mouse
occupies
MULTIPLEX PCR PROTOCOL: as noted. Lane 3.0 mm Lane 2 Lane 2
habitats
both ♦Reactions were run using puReTaq Ready-to-Go numbers to the
2.5 mm Lane 3 Lane 3
habitats PCR Beads yielding a final concentration of: agarose gel shown
•2.5 U Taq DNA polymerase in Figure 2. 2.0 mm Lane 4 Lane 4
RE-EXAMINING THE •0.2 mM dNTP
1.5 mm Lane 5 Lane 5
QUESTION: •10 mM Tris HCL
♦Our lab decided to re-examine the question using •50mM KCl
Figure 2: Varying [Mg++].
new identification techniques based on PCR •1.5 mM MgCl2 ([Mg++] was adjusted as noted
in Table 1) PCR amplified DNA from
amplified mtDNA rather morphology.
♦Both species are supposedly sympatric in ♦Primers (P.mani-F-9197, P.leuco-F-9263, and P. maniculatus in Well Set
Southeastern Wisconsin. H9375) were used at 0.5 mM. 1 and P. leucopus in Well
♦Preliminary data showed only the presence of
P. maniculatus in both arboreal and ground-layer
PCR CYCLES Set 2. See table 1 for [Mg+
♦94o C for 120 seconds +
] and lane number. Lane 6
habitats.
♦30 cycles of:
•A number of samples yielded no DNA for both Well Sets contains
•94o C for 60 seconds (Denature)
fragments when amplified by PCR. •56o C for 90 seconds (Annealing) loading dye with Xylene
♦Several possibilities could explain the lack of •72o C for 90 seconds (Polymerization) Cyanol.
P. leucopus DNA: ♦72o C for 10 minutes
•We do not have a sufficient number of ♦4o C Soak
samples.
•The PCR protocol we are using is not DNA ANALYZED:
♦2% Agarose gel in TAE
amplifying the DNA
♦Run at 100V ♦We now know that our PCR protocol is working.
• P. leucopus is not found in our area.
•Sample lanes run with Xyline Cyanol as loading ♦From here, we will work on obtaining a larger sample size
♦Solutions to these issues: dye only
•Determine whether or not the PCR protocol from local woodlots.
•Lanker lanes run with Bromophenol Blue as lead ♦If indeed we find only P. maniculatus in local woodlots, it
was working by using know tissue samples of dye) will bring into question the sympatry of the two species in
both species. Wisconsin.
•Optimize [Mg++].

Special Thanks to:

♦Dr. George Clokey for his time and patience
♦The Peromyscus Genetic Stock Center of
the University of South Carolina
♦Dr. Nathalie Tessier of the University
of Montreal, Department of Biological Science
♦The Undergraduate Research Program of
UW-Whitewater