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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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# Use 1μL of this DNA product to check concentration on the nanodrop. | # Use 1μL 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]. These instructions are for submitting a "Discount- Strip Tube order". 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. WARNING- These instructions were made specifically for sequencing PCR product, and were written by Cameron Stewart who (at the time of writing this) is a complete novice. At least skim through the linked pages. The following instructions were given to me when I asked the Sanger Sequencing staff what to do because I found the online instructions confusing. | # Prepare the solution to be Sanger Sequenced at the [https://www.biotech.wisc.edu/services/dnaseq/sequencing/sangersequencing UW-Biotech Sanger Sequencing Facility]. These instructions are for submitting a "Discount- Strip Tube order". 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. WARNING- These instructions were made specifically for sequencing PCR product, and were written by Cameron Stewart who (at the time of writing this) is a complete novice. At least skim through the linked pages. The following instructions were given to me when I asked the Sanger Sequencing staff what to do because I found the online instructions confusing. | ||
## Fill a .2ml PCR Strip Tube (not a 1.5ml Eppendorf tube) with 10ng/100bp of your pcr product (or 0.2μg/6500bp of plasmid according to [https://www.biotech.wisc.edu/services/dnaseq/sequencing/sangersequencing/guide-to-successful-sequencing Guide to successful sequencing]). For example, if your PCR product is 580bp, then you want 58ng. So, if your DNA concentration was 25ng/ | ## Fill a .2ml PCR Strip Tube (not a 1.5ml Eppendorf tube) with 10ng/100bp of your pcr product (or 0.2μg/6500bp of plasmid according to [https://www.biotech.wisc.edu/services/dnaseq/sequencing/sangersequencing/guide-to-successful-sequencing Guide to successful sequencing]). For example, if your PCR product is 580bp, then you want 58ng. So, if your DNA concentration was 25ng/μL, then you'd want to insert 58ng x 1μL/25ng = 2.32μL of your PCR product into the tube. | ||
## 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. | ||
###The [https://www.biotech.wisc.edu/services/dnaseq/sequencing/sangersequencing/guide-to-successful-sequencing Guide to successful sequencing] says "It is important to limit primer amount to 5 pmol when sequencing PCR fragments," but in person I was told to use 10pMoles. When I asked about this contradiction, they said 10 pMoles is good, but 5 pMoles is also good... >_< | ###The [https://www.biotech.wisc.edu/services/dnaseq/sequencing/sangersequencing/guide-to-successful-sequencing Guide to successful sequencing] says "It is important to limit primer amount to 5 pmol when sequencing PCR fragments," but in person I was told to use 10pMoles. When I asked about this contradiction, they said 10 pMoles is good, but 5 pMoles is also good... >_< | ||
