DNA Sequencing Protocols
Generation of PCR
Products for Sequencing
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target DNA
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300-50 pg for plasmid/simple targets
200 ng for genomic DNA
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PCR primers
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5 pmoles total
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1.25mM dntps
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2-16ul
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10x PCR buffer
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10ul
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water
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q.s. 100ul
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- HOT START
- Pre-heat a DNA thermal cycler to 95 degrees.
- Place tubes in block.
- Quickly add 2U TAQ polymerase diluted ready for use.
- Start desired run cycle, x25, linked to soak at 4 degrees.
- 95 C, 30 secs.
- 55 C, 30 secs. - depending on Tm (2-3 degrees below Tm)
- 72 C, 1 min. (P.E. 480)
- Remove excess primers and nucleotides (c-100, Quiaquick, etc). Check for product on an agarose gel.
If universal tailed primers are used and dntps are limiting, PCR product may be diluted 1/10 and sequenced with dye primers without further purification.
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