AlanFung:LabNotes/CTCF/2011-2-14: Difference between revisions
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*9. Add 100ul M-wash buffer to the column. Centrifuge at full speed for 30sec | *9. Add 100ul M-wash buffer to the column. Centrifuge at full speed for 30sec | ||
*10. Add 200ul M-Desulphonation Buffer to the column and let stand at room temperature for 20mins. After incubation centrifuge at full speed for 30sec | *10. Add 200ul M-Desulphonation Buffer to the column and let stand at room temperature for 20mins. After incubation centrifuge at full speed for 30sec | ||
*11. Add 200ul M-Wash buffer to the column. Centrfuge at full speed for | *11. Add 200ul M-Wash buffer to the column. Centrfuge at full speed for 30sec. Add another 200ul of M-wash buffer and centrifuge for an additional 30sec | ||
*12. Place the column into a 1.5ml microcentrifuge tube . Add 15ul of M-Elution Buffer directly to the column matrix. Centrifuge for 30sec at full speed to elute the DNA | *12. Place the column into a 1.5ml microcentrifuge tube . Add 15ul of M-Elution Buffer directly to the column matrix. Centrifuge for 30sec at full speed to elute the DNA | ||
===Addition of bPCR-Compatible Sites and Library Enrichment=== | |||
*1. Assemble the following reaction components at room temperature | |||
12ul Nuclease-Free water | |||
10ul bilsufite converted library | |||
25ul 2X Nextera PCR Buffer | |||
1ul 50X Nextera Primer Cocktail | |||
1ul 50X Nextera Adaptor 2 | |||
1ul Nextera MEthyl-Seq Polymerase | |||
*2. Cycle the samples in a thermocycler under the following conditions | |||
95C for 30sec | |||
25cycles of | |||
95C for 10sec | |||
60C for 10sec | |||
72C for 1min | |||
hold for 1min | |||
*3. Purify the tagged DNA using qiaquick column | |||
==Results== | ==Results== | ||
[[File:ZhangLab_2 2011-02-17 15hr 18min.jpg|500px]] | |||
*Library successfully amplified with the adapters | |||
*Band shows up around 200BP which is expected, as the transposon shears the DNA to around that size. It appears that the Meth-Seq DNA sample prep kit is working | |||
*Library preparation with Methy-Seq DNA Prep kit is successful with 100ng of starting material | |||
==Suggestions== | ==Suggestions== |
Latest revision as of 18:19, 18 February 2011
Nextera Methyl-Seq DNA Sample Prep[edit]
Objective[edit]
- Confirmation of the library prep with beta nextera methyl-seq kit with input DNA (100ng starting amount) with GM12878 gDNA.
Samples & Materials[edit]
- 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[edit]
- Tagmentation
- Purification
- Fill-in
- Purification
- Bisulfite Conversion
- Purification
- bPCR-compatible sites and library enrichment
Protocol[edit]
TagmentationTM Reaction[edit]
- 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[edit]
- 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
- 6. Add 600ul M-binding buffer to a Zymo spin column and place the column into a provided collection tube
- 7. Load the sample into the column. Close the cap and mix by inverting the column several times
- 8. Centrifuge at full speed for 30sec. Discard the flow through
- 9. Add 100ul M-wash buffer to the column. Centrifuge at full speed for 30sec
- 10. Add 200ul M-Desulphonation Buffer to the column and let stand at room temperature for 20mins. After incubation centrifuge at full speed for 30sec
- 11. Add 200ul M-Wash buffer to the column. Centrfuge at full speed for 30sec. Add another 200ul of M-wash buffer and centrifuge for an additional 30sec
- 12. Place the column into a 1.5ml microcentrifuge tube . Add 15ul of M-Elution Buffer directly to the column matrix. Centrifuge for 30sec at full speed to elute the DNA
Addition of bPCR-Compatible Sites and Library Enrichment[edit]
- 1. Assemble the following reaction components at room temperature
12ul Nuclease-Free water 10ul bilsufite converted library 25ul 2X Nextera PCR Buffer 1ul 50X Nextera Primer Cocktail 1ul 50X Nextera Adaptor 2 1ul Nextera MEthyl-Seq Polymerase
- 2. Cycle the samples in a thermocycler under the following conditions
95C for 30sec 25cycles of 95C for 10sec 60C for 10sec 72C for 1min hold for 1min
- 3. Purify the tagged DNA using qiaquick column
Results[edit]
File:ZhangLab 2 2011-02-17 15hr 18min.jpg
- Library successfully amplified with the adapters
- Band shows up around 200BP which is expected, as the transposon shears the DNA to around that size. It appears that the Meth-Seq DNA sample prep kit is working
- Library preparation with Methy-Seq DNA Prep kit is successful with 100ng of starting material