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