McClean: Sequencing Colony PCR Product: Difference between revisions
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==Protocol== | ==Protocol== | ||
# Check your colony PCR product on a gel. | # Check your colony PCR product on a gel. | ||
# If you have one clear band, follow the Zymo Research DNA Clean & Concentrator Kit instructions for cleaning up your colony PCR reaction. | # If you have one clear band, follow the Zymo Research DNA Clean & Concentrator Kit instructions for cleaning up your colony PCR reaction. | ||
# Elute your DNA (Step #5) in 10μL of H<sub>2</sub>O (NOT Elution Buffer). This is the eppendorf tube you will send for sequencing, so make sure it is clearly labeled. | ## This is a PCR reaction, so add 5 volumes of DNA Binding Buffer in Step #1. | ||
## Elute your DNA (Step #5) in 10μL of H<sub>2</sub>O (NOT Elution Buffer). This is the eppendorf tube you will send for sequencing, so make sure it is clearly labeled. | |||
# Use 2μL of this DNA product to check concentration on the nanodrop. | # Use 2μL of this DNA product to check concentration on the nanodrop. | ||
# To the 8μL of remaining DNA, add 4μL of primer at 8pmol. (Our lab oligos are at 10μM, so to get the desired 8pmol of oligo, dilute our oligos 1:5 in sterile water for a final of 2pmol/ul and add 4μL to the 8μL of DNA to be sequenced). | # To the 8μL of remaining DNA, add 4μL of primer at 8pmol. (Our lab oligos are at 10μM, so to get the desired 8pmol of oligo, dilute our oligos 1:5 in sterile water for a final of 2pmol/ul and add 4μL to the 8μL of DNA to be sequenced). | ||
==Notes== | ==Notes== | ||
Revision as of 11:25, 18 December 2015
Overview
This protocol is for sequencing the product of a yeast colony PCR.
Materials
- Colony PCR product
- DNA Clean & Concentrator Kit (Zymo Research)
- Sequencing primers
Protocol
- Check your colony PCR product on a gel.
- 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.
- 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.
- Use 2μL of this DNA product to check concentration on the nanodrop.
- To the 8μL of remaining DNA, add 4μL of primer at 8pmol. (Our lab oligos are at 10μM, so to get the desired 8pmol of oligo, dilute our oligos 1:5 in sterile water for a final of 2pmol/ul and add 4μL to the 8μL of DNA to be sequenced).
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.
References
Contact
- Megan N McClean 14:01, 20 July 2011 (EDT)
or instead, discuss this protocol.
