AlanFung:LabNotes/2015/2015-7-14: Difference between revisions

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==Swift Methyl-Seq DNA Library Prep (07/15/2015)==
==Swift Methyl-Seq DNA Library Prep (07/15/2015)==


==Denature==
==Denature (07/15/2015)==
* Transfer 15ul of bisulfite converted samples to 0.2ul PCR tube.
* Transfer 15ul of bisulfite converted samples to 0.2ul PCR tube.
* Incubate at 95C for 2 mins
* Incubate at 95C for 2 mins
* Transfer to ice for 2 mins
* Transfer to ice for 2 mins
==Adaptase==
==Adaptase==
* Prepare Master Mix
* Prepare Master Mix

Revision as of 10:04, 16 July 2015

Swift Methyl-Seq DNA Library Prep & Capturing

Summary

Experiment Person Capture Date Lib Prep Capture Conclusion
1 Noi 141104 Tang SeqCap lower complexity and higher clonal rates due to Tang et assumption the DNA fragment ends
2 Noi 141225 Kapa Hyper SeqCap lower specificity and enrichment due to capture condition
3 Alan 150115 Kapa Hyper SeqCap Mapping rates, library complexity and capture specificity is high
4 Alan 150714 Swift ? ?

Samples

  • We only have 12 reactions from the swift methyl-seq kit and Dana needs to use it for her project so I can work on 4 samples.
  • Noi made some WGBS libraries using Kapa with 1ng input (NC2, NC3, 6P2 and 6P3) on 10/24/2014
  • I am planning to capture these NC WGBS libraries with the Jun2015 probe set, together with the newly made Swift WGBS libraries on the same sample NC samples.
  • Most of the input amount used in Noi's 2nd experiment is much higher than 1ng. 6P1 and 7P8 are closer to the 1ng input.
  • After talking to Dr. Zhang we have decided to work on NC2, NC3, 6P1 and 7P8

Bisulfite Conversion using Zymo EZ DNA MEthylation-Lightning Kit (07/15/2015)

Sample Concentration ng/ul 1ng Volume ul 20ul H2O ul
NC2 0.325 3.08 16.92
NC3 0.538 1.86 18.14
6P1 0.2 5.00 15.00
7P8 0.167 5.99 14.01
  • We do not have 1ng of DNA
Sample Concentration ng/ul Volume ul 20ul H2O ul Amount Used ng
NC2 0.325 3.08 16.92 1
NC3 0.538 1.86 18.14 1
6P1 0.2 3.3 16.7 0.66
7P8 0.167 3.5 16.50 0.58
  • Add 130ul lighting conversion reagent to 20ul of sample, compensate with water if less than 20ul.
  • Vortex and centrifuge.
  • (98C 8min > 54C 60min > 10C hold)
  • Add 600ul of M-Binding Buffer to column with collection tube.
  • Load the sample to column. Close the cap and mix by inverting the column several times.
  • Centrifuge at full speed for 30s. Discard flow through.
  • Add 100 μl of M-Wash Buffer to the column. Centrifuge at full speed for 30s.
  • Add 200 μl of L-Desulphonation Buffer to the column and let stand at RT for 20 mins. After the incubation, centrifuge at full speed for 30 seconds.
  • Add 200 μl of M-Wash Buffer to the column. Centrifuge at full speed for 30 seconds.
  • Repeat this wash step.
  • Place the column into a 1.5 ml microcentrifuge tube and add 10 μl of M-Elution Buffer directly to the column matrix. Centrifuge for 30 seconds at full speed to elute the DNA.
  • The DNA is ready for immediate analysis or can be stored at or below -20°C for later use. -80C for long term storage.

Swift Methyl-Seq DNA Library Prep (07/15/2015)

Denature (07/15/2015)

  • Transfer 15ul of bisulfite converted samples to 0.2ul PCR tube.
  • Incubate at 95C for 2 mins
  • Transfer to ice for 2 mins

Adaptase

  • Prepare Master Mix
Content 1x rxn ul 4x rxn ul
Low EDTA TE 11.5 48.3
Buffer G1 4 16.8
Reagent G2 4 16.8
Reagent G3 2.5 10.5
Enzyme G4 1 4.2
Enzyme G5 1 4.2
Enzyme G6 1 4.2
Total 25 105
  • Add master mix to denatured samples vortex to mix and spin down
  • (37C 15min > 95C 2min > 10C hold)

Extension

  • Prepare Master Mix
Content 1x rxn ul 4x rxn ul
Reagent Y1 2 8.4
Enzyme Y2 42 176.4
Total 44 184.8
  • Add 44ul of MM to each PCR tube
  • Vortex spin down and run
  • (98C 1min > 62 2min > 65C 5min > 4C Hold)

Post-Extension SPRI

  • Vortex AMPure XP beads to resuspend.
  • Add 101 μl resuspended AMPure XP beads beads to the ligation reaction.
  • Mix well by pipetting up and down at least 10 times or vortex AMPure XP beads 10 seconds to resuspend.
  • Incubate for 5 minutes at room temperature.
  • Quickly spin the tube and place it on an appropriate magnetic stand to separate beads from supernatant.
  • After the solution is clear (about 5 minutes), carefully remove and discard the supernatant.
  • Be careful not to disturb the beads that contain DNA targets (Caution: do not discard beads).
  • Add 200 μl of 80% freshly prepared ethanol to the tube while in the magnetic stand.
  • Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant.
  • Repeat Step 5 once, for a total of two washes.
  • Air the dry beads for 10 minutes while the tube is on the magnetic stand with the lid open.
  • Elute the DNA target from the beads by adding 15 μl of 10 mM Tris-HCl, pH 8.0 or 0.1X TE.
  • Note: Be sure not to transfer any beads. Trace amounts of bead carry over may affect the optimal performance of the polymerase used in the PCR master mix (2X) in the subsequent PCR step.
  • Mix well by pipetting up and down, or on a vortex. After the solution is clear (about 5 minutes), carefully transfer 15 μl supernatant to a new PCR tube.

Ligation

  • Prepare master mix
Content 1x rxn ul 4x rxn ul
Buffer B1 3 12.6
Reagent B2 10 42
Enzyme B3 2 8.4
Low EDTA TE 20 84
Total 35 147
  • 25C 15min > 4C Hold

Post-Ligation SPRI

  • 1:1.2 elute with 20 ul Low EDTA TE.

Indexing PCR

  • Add 2.5ul of indexing primer to directly to the sample.
Sample Index D50X Index D7XX
NC2 502 701
NC3 502 702
6P1 502 703
7P8 502 704
  • Prepare Master Mix
Content 1x rxn ul 4x rxn ul
Low EDTA TE 10 42
Buffer R1 10 42
Reagent R2 4 16.8
Enzyme R3 1 4.2
Total 25 105
  • Add 25ul MM to each indexed sample.
  • Vortex and spin down.
  • 98C 30sec > 12x (98C 10sec > 60C 30sec > 68C 60sec) > 4C Hold.

Post-PCR SPRI

  • (1:0.8) 40ul beads elute with 20ul Low EDTA TE.

File:ZhangLab 2 2015-07-15 23hr 17min.jpg