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McClean: Sequencing Colony PCR Product: Difference between revisions
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McClean: Sequencing Colony PCR Product (view source)
Revision as of 11:25, 18 December 2015
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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== | ||
