sequencing pcr product or plasmid afrer clonning?

[DanielT]

Hi… I am new in the sequencing and still confuse what method should I use. I want to sequence the gene as in data base. We design primer sets for these sequence and amplify it by pcr. I am now would like to check if I got the right gene and would like to sequence it. My collegue suggest me to clone the pcr product to pGEm then sequence the colonies (plasmid dna from the colony). WHy I should do the clonning before sequencing and can I do direct sequencing from my PCR product? What will make the results different if I do directly from PCR product or clonning?

Thanks….

[Daniel]

Hi Debbie

You can sequence the PCR product directly – this is generally much simpler than cloning and sequencing. The only thing you need to do first is remove the leftover primers and dNTP. I would recommend using a PCR product purification kit (there are hundreds out there) to do this. Just make sure that you don’t use too much of the PCR product in the sequencing reaction (ie follow the bigdye kit instructions).

Cheers

Daniel Tillett
Nucleics Support

[Author]

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