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.