AlanFung:LabNotes/CTCF/2011-2-14

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Revision as of 21:18, 15 February 2011 by >Alan6017518 (→‎Protocol)
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Nextera Methyl-Seq DNA Sample Prep

Objective

  • Confirmation of the library prep with beta nextera methyl-seq kit with input DNA (100ng starting amount) with GM12878 gDNA.

Samples & Materials

  • Nextera Sample Prep Kit_Epicentre_NMS072910B
NexteraTM Methyl-Seq Tagmentation Mix (Illumina-compatible) 
5X Nextera Reaction Buffer (HMW)
50X Nextera Primer Cocktail (Illumina-compatible) 
50X Nextera Adaptor 2 (Illumina-compatible) 
2X Nextera Fill-in Buffer 
2X Nextera PCR Buffer 
200X Nextera Read 1 Primer 
200X Nextera Read 2 Primer 
200X Nextera Index Read Primer  
Nextera Methyl-Seq Polymerase 
  • Input DNA
  • Bisulfite conversion kit
  • Qiagen Purification Kit

Overview

  • Tagmentation
  • Purification
  • Fill-in
  • Purification
  • Bisulfite Conversion
  • Purification
  • bPCR-compatible sites and library enrichment

Protocol

  • A. TagmentationTM Reaction
  • 1. Prior to assembling the reaction, briefly centrifuge ALL tubes to assure that the reagents are at the bottom of the tubes.
  • 2. Assemble the following reaction components on ice, in the order listed:
xul         Nuclease-Free Water 
2ul        100ng   Target DNA
4ul         5X Nextera HMW Reaction Buffer 
1ul         Nextera Methyl-Seq Tagmentation Mix (Illumina-compatible)
20ul       Total reaction volume 
  • 3. Mix briefly by vortexing, and incubate at 55C for 5 minutes.
  • 4. Purify the tagmented DNA using Qigen column and elute with 11ul Elute buffer
-The final eluted volume should be ~10 μl. 5.	Add 10 μl of 2X Nextera Fill-in Buffer and 1 μl of Nextera Methyl-Seq Polymerase to 10 μl of the
eluted/Tagmented DNA. Mix and incubate at 70oC for 10 minutes. 6.	Purify the Fill-in reaction using a Zymo DNA Clean & Concentrator-5 Kit, as in Step 4 above.

Results

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