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McClean: Sequencing Colony PCR Product: Difference between revisions
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McClean: Sequencing Colony PCR Product (view source)
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# 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. | ||
## This is a PCR reaction, so add 5 volumes of DNA Binding Buffer in Step #1. | ## 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). | ## Elute your DNA (Step #5) in 10μL of H<sub>2</sub>O (NOT Elution Buffer). | ||
# Use 2μL of this DNA product to check concentration on the nanodrop. | # Use 2μL of this DNA product to check concentration on the nanodrop. | ||
# Prepare the solution to be Sanger Sequenced at the [https://www.biotech.wisc.edu/services/dnaseq/sequencing/sangersequencing UW-Biotech Sanger Sequencing Facility]. The [https://www.biotech.wisc.edu/services/dnaseq/sequencing/sangersequencing/sanger-submission Sanger Submission], the [https://www.biotech.wisc.edu/services/dnaseq/sequencing/sangersequencing/guide-to-successful-sequencing Guide to successful sequencing], and the [https://www.biotech.wisc.edu/services/dnaseq/sequencing/sangersequencing/sequencing-materials-and-methods Materials and methods] pages may be helpful. | # Prepare the solution to be Sanger Sequenced at the [https://www.biotech.wisc.edu/services/dnaseq/sequencing/sangersequencing UW-Biotech Sanger Sequencing Facility]. The [https://www.biotech.wisc.edu/services/dnaseq/sequencing/sangersequencing/sanger-submission Sanger Submission], the [https://www.biotech.wisc.edu/services/dnaseq/sequencing/sangersequencing/guide-to-successful-sequencing Guide to successful sequencing], and the [https://www.biotech.wisc.edu/services/dnaseq/sequencing/sangersequencing/sequencing-materials-and-methods Materials and methods] pages may be helpful. | ||
## Fill a .2ml PCR Strip Tube (not a 1.5ml Eppendorf tube) with 10ng/100bp of your pcr product. For example, if your DNA product is 580bp, then you want 58ng. So, if your DNA concentration was 25ng/μMol, then you'd want to insert 58ng x 1μL/25ng = 2.32μL of your PCRed DNA into the tube) solutio product, 10pMol (10<sup>-12</sup> moles) of primer, and bring the entire volume to 20μL with DI water. | ## Fill a .2ml PCR Strip Tube (not a 1.5ml Eppendorf tube) with 10ng/100bp of your pcr product. For example, if your DNA product is 580bp, then you want 58ng. So, if your DNA concentration was 25ng/μMol, then you'd want to insert 58ng x 1μL/25ng = 2.32μL of your PCRed DNA into the tube) solutio product, 10pMol (10<sup>-12</sup> moles) of primer, and bring the entire volume to 20μL with DI water. | ||
## Fill the tube with 10 picoMoles of one primer. For example, if your primer is at 10μM concentration, then insert 10 x 10<sup>-12</sup> Moles * (1L/10*10<sup>-6</sup>moles)=10<sup>-6</sup>L=1μL. | ## Fill the tube with 10 picoMoles of one primer. For example, if your primer is at 10μM concentration, then insert 10 x 10<sup>-12</sup> Moles * (1L/10*10<sup>-6</sup>moles)=10<sup>-6</sup>L=1μL. | ||
## Label the strip tubes individually. Then wrap them in foil and on a peice of tape on the foil write your PI's name, your name, the date, and the name of the set of samples. (The foil itself isn't special, a bag could be used too, anything to keep them together and labelled.) | |||
## If you are still confused, the people at the facility are extremely patient and helpful. Talking to them in person may be best. | ## If you are still confused, the people at the facility are extremely patient and helpful. Talking to them in person may be best. | ||
==Notes== | ==Notes== | ||
