Yeast colonies are suspended in complete PCR buffer and transferred to a thermal cycler for 35 cycles of PCR. The products of the amplification reaction are analyzed by gel electrophoresis. - [Read Analyzing Yeast Colonies by PCR Protocol]
In the first part of this protocol, the linear range of amplification is determined by carrying out 10 identical PCRs in the presence of [{alpha}-32P]dCTP and stopping one reaction after every two cycles. Amplification products are quantified on a denaturing polyacrylamide gel and the results plotted on a graph (counts per minute vs. cycle number). Total RNA is used as an internal control. - [Read Relative RT-PCR: Determining the Linear Range of Amplification and Optimizing the Primers:Competimer]
Alfalfa is an outcrossing species, cultivars consist of populations rather than individual homozygous or
inbred lines. After only two cycles of self-pollination, severe inbreeding depression eliminates selfed
individuals from populations. To obtain sufficient plant material of relative genetically uniformity (these
plants will still necessarily be heterozygous) for experimental purposes, it may be necessary to propagate a
single individual or clone. - [Read Vegetative Propagation of Alfalfa by Stem Cuttings]