Help on PCR details

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racerwoman
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Help on PCR details

Post by racerwoman » Tue Apr 25, 2006 9:11 pm

hey,

I have a question for someone who understands PCR, etc.

I've never done PCR and I have to do a presentation on a paper that did some PCR. The paper I'm reading says, "Aberrant migrating amplicons were excised from the gel, reamplified using Pfu DNA polymerase, cloned into Eco-RV-digested pZeRo, and sequenced using M13 primers and big-dye terminators."

I understand everything until "Eco-RV..." I'm guessing that pZeRo is a plasmid and that Eco-RV is the restriction enzyme, but I dont understand the role of the M13 primers and what big-dye terminators are. I tried looking it up on various websites but it's just not making any sense. If someone could help, I'd really appreciate it. Thanks!

RW

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canalon
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Post by canalon » Tue Apr 25, 2006 9:58 pm

You guessed right for EcoR V and pZeRo.
The M13 primers are common primers for sequencing, because many commercial plasmid includes the complementary sequence around the insertion site. So you don't need to know the sequence of your insert to design sequencing primers. Big-Dye is one of the kind of reagents used for automated sequencing. The most common one, nothing special. But sometimes other reagents are used (for example in case of GC rich sequences).

Hope this helps
Patrick

Science has proof without any certainty. Creationists have certainty without
any proof. (Ashley Montague)

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