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archive:lab_protocols:solid_run1 [2010/04/06 17:41]
hyjkim
archive:lab_protocols:solid_run1 [2015/09/02 16:57] (current)
ceisenhart ↷ Page moved from lab_protocols:solid_run1 to archive:lab_protocols:solid_run1
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 ===== Solid Sequencing Run 1 ===== ===== Solid Sequencing Run 1 =====
- 
 ==== Meta ==== ==== Meta ====
 +
 +Prepared by: Eveline Hesson
 +
 Date: Date:
  
 Overview: Overview:
 +
 ==== DNA Extraction ==== ==== DNA Extraction ====
 ==== Library Preparation ==== ==== Library Preparation ====
 +
 +This library was prepared by following ABI's standard 2x25 mate-pair library preparation protocol. {{:​lab_protocols:​solid3_libprep_guide.pdf|}}
 +
 +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.
- 
-^ 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 Kit217 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 | +=== Purify the End-Repaired DNA using the QIAquick Gel Extraction Kit === 
-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. 
  
 ==== Emulsion PCR ==== ==== Emulsion PCR ====
  
 ==== Sequencing Data ==== ==== Sequencing Data ====
archive/lab_protocols/solid_run1.1270575684.txt.gz · Last modified: 2010/04/06 17:41 by hyjkim