archive:lab_protocols:solid_run1
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archive:lab_protocols:solid_run1 [2010/04/06 17:53] hyjkim |
archive:lab_protocols:solid_run1 [2010/04/07 17:21] hyjkim |
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| ===== Solid Sequencing Run 1 ===== | ===== Solid Sequencing Run 1 ===== | ||
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| ==== Meta ==== | ==== Meta ==== | ||
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| + | Prepared by: Eveline Hesson | ||
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| Date: | Date: | ||
| Overview: | Overview: | ||
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| ==== DNA Extraction ==== | ==== DNA Extraction ==== | ||
| ==== Library Preparation ==== | ==== Library Preparation ==== | ||
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| + | This library was prepared by following ABI's standard 2x25 mate-pair library preparation protocol. {{:lab_protocols:solid3_libprep_guide.pdf|}} | ||
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| + | Detailed notes of the protocol can be found in the following pdf: {{:lab_protocols:solid-mate-pair-notes.pdf|}} | ||
| === Shearing DNA === | === Shearing DNA === | ||
| Extracted genomic DNA was sheared using a [[https://products.appliedbiosystems.com/ab/en/US/adirect/ab?cmd=catNavigate2&catID=604432|Hydroshear]] to an approximate size of 4-5kb. The shearing was done using Standard_Assembly, sc 15, 5 cycles for 150 µl total volume. | Extracted genomic DNA was sheared using a [[https://products.appliedbiosystems.com/ab/en/US/adirect/ab?cmd=catNavigate2&catID=604432|Hydroshear]] to an approximate size of 4-5kb. The shearing was done using Standard_Assembly, sc 15, 5 cycles for 150 µl total volume. | ||
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| - | ^ Component^ ^ Tube 1 ^ Tube 2 ^ | ||
| - | |DNA (µg) | 15 | 15 | | ||
| - | |DNA (µL) | 59 | 59 | | ||
| - | |Nuclease Free Water (µl) | 66 | 66 | | ||
| - | |Total | 125 | 125 | | ||
| Total recovered DNA after hydroshear was 217 µl or 26 µg. | Total recovered DNA after hydroshear was 217 µl or 26 µg. | ||
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| === Repair the DNA Ends === | === Repair the DNA Ends === | ||
| - | ^ Component ^ Volume (µL) ^ Lot Number ^ | + | The DNA ends of sheared gDNA were blunt ended and phosphorylated using Epicentre's End-It DNA repair Kit. 217 µl of sheared gDNA was reacted in a total volume of 400 µl according to manufacturer's standards. The reaction was incubated for 30 minutes at room temperature. |
| - | | Sheared DNA | 217 | E85-81001 | | + | |
| - | | 10x End-it Buffer | 40 | E85-81001 | | + | |
| - | | End-It ATP (10 mM) | 40 | E85-81001 | | + | |
| - | | End-It dNTPs (2.5 mM) | 40 | E85-81001 | | + | |
| - | | End-It Enzyme Mix | 12 | E85-81001 | | + | |
| - | | Nuclease-free water | 51 | 0903130 | | + | |
| - | | Total | 400| - | | + | |
| - | + | ||
| - | Incubate the mixture at room temperature for 30 minutes. | + | |
| === Purify the End-Repaired DNA using the QIAquick Gel Extraction Kit === | === Purify the End-Repaired DNA using the QIAquick Gel Extraction Kit === | ||
archive/lab_protocols/solid_run1.txt · Last modified: 2015/09/02 16:57 by ceisenhart
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