PCR Preparation of a lacZ Probe

A digoxigenin (DIG)-labeled probe is generated using the PCR reaction and the following reagents:

1. The PCR mix should be prepared as follows:

1 |g Plasmid DNA (does not need to be linearized) 2 |L

10X Taq buffer (Boehringer Mannheim 1277049, 5 |L

Indianapolis, IN) 10 mM dNTP (see Subheading 2.1., item 2) 1 |L

Primer E128 (lacZ inner 5' primer position 711-730

Primer E129 (lacZ inner 3' primer position 1140-1159

Water 35 |L

Paraffin oil 100 |L

2. The 10-mM dNTP mix is made up of the following nucleotides (Boehringer Mannheim 1277049):

6.5 mM DIG-11-dUTP (Boehringer Mannheim 1093088: alkali stock).

3. Taq DNA polymerase (Boehringer Mannheim 1435094).

4. Variable-temperature PCR unit (Omingene, Hybaid, Holbrook, NY).

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