McClean: Sequencing Colony PCR Product

Overview

This protocol is for sequencing the product of a yeast colony PCR.

Materials

• Colony PCR product
• DNA Clean & Concentrator (Zymo Research)
• Sequencing primers

Protocol

1. Check your colony PCR product on a gel.
2. If you have one clear band, follow the Zymo Research DNA Clean & Concentrator Kit instructions for cleaning up your colony PCR reaction. (This is a PCR reaction, so add 5 volumes of DNA Binding Buffer in Step #1.)
3. Elute your DNA (Step #5) in 10μL of H₂O (NOT Elution Buffer). This is the eppendorf tube you will send for sequencing, so make sure it is clearly labeled.
4. Use 2μL of this DNA product to check concentration on the nanodrop.
5. To the 8μL of remaining DNA, add 4μL of primer.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

• Megan N McClean 15:25, 25 March 2013 (EDT) Any method of cleaning up the DNA should be fine. In my hands, the Zymo Research kit seems to work the best. Make sure to elute in WATER and NOT elution buffer in the final step. This seems to help a lot.

Contact

• Megan N McClean 14:01, 20 July 2011 (EDT)

or instead, discuss this protocol.