PCR 2

Learning goals

You will add DNA adaptors to the ends of the PCR product from PCR1. These adaptors prepare the DNA for sequencing.

By the end of this module, you should be able to:

• explain why we are adding adaptors to the ends of the PCR product from PCR1

• explain what an index is and why we need them

• explain how we will add adaptors to the ends of our PCR1 product

Safety Information and MSDS

• Everything in this lab is safe, and may be disposed of in the garbage or drain.

• Keep all chemicals away from food and food preparation equipment.

• Work on the provided bench paper to absorb any spills.

• Centrifuge: Always balance your centrifuge, and wait until the centrifuge is done spinning before opening the lid.

• Wash hands and clean counter tops when you are done.

• MSDS: If you click through this link, you will find Material Safety Data Sheets (MSDS) for chemicals used in this lab.

Reading: What does PCR2 do?

Read this document to learn about the adaptors we are adding to the ends of the PCR product from PCR1, why we are adding them, and how we will add them.

Assignment: What does PCR2 do?

Test your understanding of PCR2 by answering these questions

Preparing to use your miniPCR thermocycler

In case you need a reminder of how to program your thermocycler, check here.

Here's what you should program for PCR2:

1) 95˚C 3 minutes Denature template

2) 98˚C 20 seconds Denature template

3) 60˚C 15 seconds Primers Anneal to template

4) 72˚C 15 seconds This is the optimal temperature for polymerase activity, so new DNA is synthesized

5) go to step 2 and repeat 7 times (total of 8 cycles) Synthesize new copies of DNA

6) 72˚C 1 minute This allows any strands that may not have been synthesized to completion to finish extension

Assignment:

Let your instructor know which PCR2 primers you used with which herbal supplement sample. 

We need this information for sequence data analysis.

Protocol: PCR2

Read this to learn how to set up PCR2.

Be sure to read and understand all instructions before beginning

Assignment: PCR2 photos

Submit a photo or photos showing that you did PCR2