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Easily Amplify Genomic DNA with Long-Distance PCR

94C for 30 seconds, 55C for 30 seconds, and 72C for 2, 4, or 6 minutes to amplify 2, 4, or 6 kb DNA fragments, respectively (1 minute per ~1 kb of template) (see Table 1). Following amplification, 5-l aliquots of each reaction mixture were analyzed by electrophoresis in 0.5X TBE 0.9% agarose gels, and the amplification products were visualized by staining with ethidium bromide.

Table 1
Conditions Used to Amplify Mouse SMHC Gene Fragments with Specific Primers

The Amplified Fragment

Specific Primers

Stratagene 10X TaqPlus Long
PCR Buffer Used

Time at 72C
(Extension Step) (Minutes)

Size of the Fragment (kb)

Upstream

Downstream

#1

Ex5aFOR

Ex5bREV

low salt

2

2.1

#2

Ex5bFOR

Ex6REV

low salt

'"/>

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