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