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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 12:33, 18 December 2015
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# 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. For example, if your PCR 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 PCR product into the tube. | ## Fill a .2ml PCR Strip Tube (not a 1.5ml Eppendorf tube) with 10ng/100bp of your pcr product. For example, if your PCR 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 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. 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... >_< | ## 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... >_< | |||
## Bring the total volume to 20μL with DI water. For example, if your DNA + primer volume is 3.3μL, then insert 16.7μL of water. | ## Bring the total volume to 20μL with DI water. For example, if your DNA + primer volume is 3.3μL, then insert 16.7μL of water. | ||
## 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.) | ## 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.) | ||
