Inter simple sequence repeats (ISSR) uses microsatellite sequences as primers in PCR to generate multilocus markers that vary between organisms. Analysis and comparison of ISSR’s between organisms can reveal relatedness

Protocol:

Used ISSR markers (OMAR – (GAG)^4 RC) and psbA

On Samples (Left->Right)

GWC01 , CRA05 , PRO01 , PFR01 ,PRM01

20 tube Master mix for ISS (1 microliter template per tube)

ddH20 -> 250 microliters

10x buffer + Mg -> 60 microliters

BSA -> 20 microliters

dNTPs ->40 microliters

OMAR Primer -> 5 microliters

Taq -> 5 microliters

20 tube Master mix for psbA with following volumes in each tube

ddH20 -> 15.4 microliters

10x buffer + Mg -> 2 microliters

BSA -> 1 microliters

dNTPs ->.20 microliters

F Primer -> .2 microliters

R Primer -> .2 microliters

Taq -> .04 microliters

Placed on thermocycler