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Amplification of Mouse cDNAs for Microarrays Using the Eppendorf ,,, MasterTaq Kit

MasterTaq) amplified all targets specifically, it was then investigated as to whether or not a dilution of the enzyme would still result in successful amplification from an overnight culture. The enzyme was tested at full strength (7.5 U/150 l reaction) and at half strength (3.25 U/150 l reaction). We observed that a one-half strength dilution of the enzyme yielded successful amplification of DNA from bacterial clones (Fig. 2).


Figure 2
Comparison of Eppendorf MasterTaq at half-strength and full-strength concentrations in amplifying 96-well plate overnight cultures of mouse clones.

Amplification of the mouse unigene set using the Eppendorf MasterTaq Kit

The Eppendorf MasterTaq Kit was used to amplify all ~8 K targets that comprise the mouse Unigene set. In total 8,734 amplifications were performed. Figure 2 shows agarose gel analyses of representative mouse cDNA amplifications using the Eppendorf MasterTaq kit in a 96-well plate format. In general, strong unique amplicons were observed. The results were classified as follows:

~ 7,997 single amplicons (strong bands)
~ 359 single amplicons (weak bands)
~ 293 multiple bands (typically two or more)
~ 85 failures

The cDNA clones that generated multiple bands were re-racked and amplified with a nested primer set (data not shown). For the most part, multiple bands were still observed indicating that these cDNAs represent mixed clones. This signifies ~3.4% contamination, typically found in amplification of a large clone set.

Amplicon Yield Per 150 l Reaction

Source Plate N
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