Noi/NOTES/2013-5-22

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Adaptor ligation of CoRE fragmented DNA (Eric's May 21, 2013 experiment)[edit]

  • Got 16 samples from Eric for ligation using NEBNext adaptor. (Samples were already in 0.2 ml PCR Tube Strips, low-Profile tube (Biorad))
  • MDA and CoRE fragmentation were performed by Eric and documented on Eric's note on May21, 2013: [[1]]
  • Note that the order of samples in 8-striptube ordered by Eric and me was in the opposite way within the striptube. So number 1-8 by Eric will be flipped to 8-1 by me and also for 19-16 --> 16-9.

Adaptor ligation[edit]

Reaction set up

Components Volume (ul)
Fragmented DNA 6.00
1.5uM NEBNext adaptor 1.67
Ligation reaction mix 42.33
Total 50.00

NEBNext adaptor preparation

  • I used 1.5uM adaptor prepared in the previous experiment
  • I diluted 15uM adaptor to 1.5uM (mix 10ul of 15uM NEBNext adaptor with 90ul H2O) and aliquoted 12ul into 8-striptubes and saved at -20C
  • The concentration of adaptor for ultra-low input DNA (<=10ng) in 50ul reaction is 50nM

Ligation reaction mix

Ligation reaction mix 1x rxn 16x rxn mix
2X KAPA Rapid Ligation Buffer 25.00 400.00
KAPA Rapid T4 DNA Ligase 2.00 32.00
H2O 15.33 245.28
Total 42.33 677.28
  • Aliquot 42.33 ul of ligation reaction mix to each tube containing 6ul of fragmented DNA
  • Add 1.67ul of 1.5uM NebNext loob adaptor. Mix well by pipetting 15-20 times and quick spin.
  • 25C for 10min
  • Add 2ul USER. Mix well by pipetting 15-20 times and quick spin. 37C for 15min
    • For the library prep using NEBNext® DNA Library and this loop adaptor, they used 3ul USER in total volume 50ul or 83.5ul, 37 C for 15 min. I used a little less

Bead purification

  • 50ul AmPure beads mix by pipetting 10 times
  • Wait 10min
  • Sit on magnetic stand 5min
  • Wash twice with freshly prepared 80% EtOH (after adding 80% EtOH, I wait for 30sec before pipette up and down few times and discard spnt)
    • I wash with 160ul of 80% EtOH since I used low profile tube.
  • Dry the beads for 3 min
  • Eluted with 20ul H2O (resuspend and wait for 2min, used all for PCR)

Amplification[edit]

Components 1x rxn 16.5 rxn mix
adapter ligated DNA 20.00 0.00
PCR_F (NEB, 10uM) 1.00 16.50
NEBNext.IndX (NEB, 10uM) 1.00 0.00
2X KAPA SYBR MM 25.00 412.50
H2O 3.00 49.50
Total volume 50.00 478.50
  • Aliquot 29ul, add 1ul of NEBNext Indx, 20ul of adapter ligated DNA
Samples Indx Samples Indx
#1 NEBNext Index1 #9 NEBNext Index1
#2 NEBNext Index2 #10 NEBNext Index2
#3 NEBNext Index3 #11 NEBNext Index3
#4 NEBNext Index4 #12 NEBNext Index4
#5 NEBNext Index5 #13 NEBNext Index5
#6 NEBNext Index6 #14 NEBNext Index6
#7 NEBNext Index7 #15 NEBNext Index7
#8 NEBNext Index8 #16 NEBNext Index8
Program (Eppendorf Realplex)

98C - 30s, (98C - 10s, 65C - 30s, 72C - 45s)x15, 72C - 2min, hold at 15C

I stopped the reaction at 12 cycles
File:20130522 qPCR CoRE.png
  • Eric should provide the sample info.
  • PAGE analysis to verify the PCR products by loading 5 ul of PCR product without bead purification (1/10 of total volume)
File:ZhangLab 2 2013-05-22 13hr 33min gel1 1-8.jpg  File:ZhangLab 2 2013-05-22 13hr 35min gel2 9-18.jpg
  • Work for all samples, but the library size was a little high (average ~800bp) than the library got from Jeff's experiment in April30, 2013.

2013_05_23

  • Dr. Zhang suggested to pool Index1-8 (two pools because of the index duplicates) together, size-select 300-700bp fragments, and do 5 million SE36bp reads per pool

PAGE quantification and sequencing library pooling

  • I did PAGE quantification on the gel images above (300-700bp range) and pooled 75ng each library.
  • Note, I din't do bead purification before PAGE size-selection. However, if libraries needed to be saved for future experiment, I would do bead purify.

Pool1: #1 - #8

Sample # Index Conc. (ng/ul) Yields in 45ul (ng) Volume for 75ng (ul)
#1 Index 1 8.51 383.13 8.81
#2 Index 2 9.05 407.32 8.29
#3 Index 3 3.86 173.74 19.43
#4 Index 4 5.12 230.32 14.65
#5 Index 5 6.96 313.37 10.77
#6 Index 6 7.52 338.38 9.97
#7 Index 7 9.05 407.12 8.29
#8 Index 8 6.72 302.42 11.16

Pool2: #9 - #16

Sample # Index Conc. (ng/ul) Yields in 45ul (ng) Volume for 75ng (ul)
#9 Index 1 5.80 260.82 12.94
#10 Index 2 5.93 266.90 12.65
#11 Index 3 8.04 361.67 9.33
#12 Index 4 7.12 320.47 10.53
#13 Index 5 4.42 199.01 16.96
#14 Index 6 8.48 381.72 8.84
#15 Index 7 6.84 307.85 10.96
#16 Index 8 7.76 349.40 9.66

PAGE size-selection

  • Each pool has ~600ng of 300-700bp fragments
  • Each pool purified in 1 of 6% 5-well TBE gel
File:ZhangLab 2 2013-05-23 13hr 47min SS-pool1 e.jpg  File:ZhangLab 2 2013-05-23 13hr 50min aa-pool2.jpg
  • Resuspend each pool with total 30ul H2O

PAGE verification of sequencing libraries

  • Loaded 1.5ul of each library pool in 6% TBE gel
File:ZhangLab 2 2013-05-23 19hr 13min EC-MEF-PAGE.jpg

P1 = EC-MEF-Dev7-1-8-May21-2013
P2 = EC-MEF-Dev7-9-16-May21-2013
*** It seems like I cut the gel too close to 800bp especially library pool2 
*** To cut the size precisely, may need to change DNA ladder like 100bp DNA ladder to locate the 300bp and 700bp fragments

Library ID, Pool1: EC-MEF-Dev7-1-8-May21-2013 : conc. by Qubit dsDNA HS (used 2ul for assay) = 3.20 ng/ul or 9.84nM
Library ID, Pool2: EC-MEF-Dev7-9-16-May21-2013 : conc. by Qubit dsDNA HS (used 2ul for assay) = 4.60 ng/ul or 14.15nM

  • MW of 500bp (average of 300-700bp) = 500bp * 650Da/bp + 159Da ~ 325159Da or g/mole
  • Alan did qPCR to quantify the concentration of the libraries again

GAII run: HL153 (SE)

  • EC-MEF-Dev7-1-8-May21-2013: Lane 1
  • EC-MEF-Dev7-9-16-May21-2013: Lane 2