♦we now know that our pcr protocol is working. ♦from here, we will work on obtaining a larger...

♦We now know that our PCR protocol is working.♦From here, we will work on obtaining a larger sample size from local woodlots.♦If indeed we find only P. maniculatus in local woodlots, it will bring into question the sympatry of the two species in Wisconsin.

[Mg++ ] Well Set 1 Well Set 2

3.5 mm Lane 1 Lane 1

3.0 mm Lane 2 Lane 2

2.5 mm Lane 3 Lane 3

2.0 mm Lane 4 Lane 4

1.5 mm Lane 5 Lane 5

♦Tail tissue samples of P. maniculatus and P. leucopus were obtained from the Peromyscus Genetic Stock Center.♦Approximately 3 mm of tissue were clipped from each tail.

DNA PREP:♦Samples were ground on ice in 0.5 ml of normal saline using a micro-pestle in a 1.5 ml microfuge tube♦0.5 ml 10% Chelex was added♦Samples were incubated in a boiling water bath for 10 minutes♦Tissue debris and Chelex were pelleted by a 30 second spin down in an Eppendorf microcentrifuge at 14,000 rpm♦The supernatant (ca 0.5 ml) was saved and the pellet discarded.

MULTIPLEX PCR PROTOCOL: ♦Reactions were run using puReTaq Ready-to-Go PCR Beads yielding a final concentration of:

•2.5 U Taq DNA polymerase•0.2 mM dNTP•10 mM Tris HCL•50mM KCl•1.5 mM MgCl2 ([Mg++] was adjusted as noted in Table 1)

♦Primers (P.mani-F-9197, P.leuco-F-9263, and H9375) were used at 0.5 mM.

PCR CYCLES♦94o C for 120 seconds♦30 cycles of:

•94o C for 60 seconds (Denature)•56o C for 90 seconds (Annealing)•72o C for 90 seconds (Polymerization)

♦72o C for 10 minutes ♦4o C Soak

DNA ANALYZED:♦2% Agarose gel in TAE ♦Run at 100V

•Sample lanes run with Xyline Cyanol as loading dye only•Lanker lanes run with Bromophenol Blue as lead dye)

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

Figure 1: DNA Analysis.

PCR amplified DNA from

P. leucopus and P.

maniculatus tissue were run as

described. Lanes 1 & 6,

Loading dye with Xylene

Cyanol and Bromophenol

blue; lanes 2 & 5, pGEM

DNA markers (Promega,

Madison, WI); lane 3, P.

leucopus PCR amplified DNA

and lane 4, P. maniculatus

PCR amplified DNA.

Figure 2: Varying [Mg++].

PCR amplified DNA from

P. maniculatus in Well Set

1 and P. leucopus in Well

Set 2. See table 1 for [Mg+

+] and lane number. Lane 6

for both Well Sets contains

loading dye with Xylene

Cyanol.

Table 1: [Mg++] for the PCR reactions are as noted. Lane numbers to the agarose gel shown in Figure 2.

RESOURCE PARTITIONING:♦A strategy developed to reduce competition for resources between two species with similar ecologic niches that occupy the same area.♦Previous studies suggest that resource partitioning

does occur between Peromyscus maniculatus (deer mouse), and Peromyscus leucopus (white-footed mouse).

•Based on tail morphology which can be problematic due to the two species being almost identical (see images below).

RE-EXAMINING THE QUESTION:

♦Our lab decided to re-examine the question using new identification techniques based on PCR amplified mtDNA rather morphology.♦Both species are supposedly sympatric in Southeastern Wisconsin.♦Preliminary data showed only the presence of P. maniculatus in both arboreal and ground-layer habitats.

•A number of samples yielded no DNA fragments when amplified by PCR.

♦Several possibilities could explain the lack of P. leucopus DNA:

•We do not have a sufficient number of samples.•The PCR protocol we are using is not amplifying the DNA• P. leucopus is not found in our area.

♦Solutions to these issues:•Determine whether or not the PCR protocol was working by using know tissue samples of both species.•Optimize [Mg++].

Range of P. maniculatus: mouse occupies both habitats

Range of P. leucopus: mouse occupies both habitats

Overlapping Range: mice show resource partitioning with P. maniculatus occupying the trees and P.

leucopus occupying the ground

Serenity Mutchler

Kara Kamps

SimonSchmidt

Mentor: Dr. George Clokey