AlanFung:LabNotes/CTCF/2011-2-14: Difference between revisions

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  Mix and incubate at 70C for 10 minutes.  
  Mix and incubate at 70C for 10 minutes.  
*6. Purify the Fill-in reaction using a Qiagen column as in Step 4 above.
*6. Purify the Fill-in reaction using a Qiagen column as in Step 4 above.
===Bisulfite Conversion of Tagged Nextera Fragments===
*1. Add 900ul water, 300ul of M-Dilution Buffer, and 50ul M-Dissolving buffer to a tube of CT Conversion Reagent
*2. Mix at room temperature with frequent vortexing or shaking for 10 minutes
*3. Preparation of M-Wash buffer
*4. Mix 10ul nuclease free water with 130ul of the CT Conversion Reagent to 10ul of your DNA sample in a PCR tube
*5. Place the sample tube in a thermal cycler and perform the following steps
98C for 10mins
64C for 2.5hrs
4C forever
Following bisulfite conversion the DNA fragments should be purified and eluted in a volume of 10 to 15 μl. Make sure the DNA is desulfonated before proceeding to the next step.


==Results==
==Results==
==Suggestions==
==Suggestions==

Revision as of 18:27, 16 February 2011

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

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:
12.67ul Nuclease-Free Water 
2.33ul   100ng NA12878 gDNA (42.9ng/ul nanodrop check on 2/15/11)
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
  • 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 70C for 10 minutes. 
  • 6. Purify the Fill-in reaction using a Qiagen column as in Step 4 above.

Bisulfite Conversion of Tagged Nextera Fragments

  • 1. Add 900ul water, 300ul of M-Dilution Buffer, and 50ul M-Dissolving buffer to a tube of CT Conversion Reagent
  • 2. Mix at room temperature with frequent vortexing or shaking for 10 minutes
  • 3. Preparation of M-Wash buffer
  • 4. Mix 10ul nuclease free water with 130ul of the CT Conversion Reagent to 10ul of your DNA sample in a PCR tube
  • 5. Place the sample tube in a thermal cycler and perform the following steps
98C for 10mins
64C for 2.5hrs
4C forever


Following bisulfite conversion the DNA fragments should be purified and eluted in a volume of 10 to 15 μl. Make sure the DNA is desulfonated before proceeding to the next step.

Results

Suggestions