Noi/NOTES/2013-5-24: Difference between revisions
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* Got 24 samples from Eric for ligation using NEBNext adaptor. | * Got 24 samples from Eric for ligation using NEBNext adaptor. | ||
* MDA and CoRE fragmentation were performed by Eric and documented on Eric's note on May23, 2013: [[http://genome-tech.ucsd.edu/LabNotes/index.php/EricChu:LabNotesMDA/2013-5-23]] | * MDA and CoRE fragmentation were performed by Eric and documented on Eric's note on May23, 2013: [[http://genome-tech.ucsd.edu/LabNotes/index.php/EricChu:LabNotesMDA/2013-5-23]] | ||
* 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 17-24 --> 24-17. | |||
* All procedures of adaptor ligation and amplification were the same as my experiment on May22, 2013 | * All procedures of adaptor ligation and amplification were the same as my experiment on May22, 2013 | ||
== Adaptor ligation == | == Adaptor ligation == | ||
Line 112: | Line 113: | ||
* All samples worked as expected since Eric did qPCR to verify the yield of MDA. | * All samples worked as expected since Eric did qPCR to verify the yield of MDA. | ||
* The average size of libraries looked smaller than the ones on May22 | * The average size of libraries looked smaller than the ones on May22 | ||
* This time, I reduced the number of cycle from 12 cycles to 11 cycles. Next time with the same amount of input, I may reduce to 9 or 10 cycles to reduce bias from amplification. | |||
<br> | |||
''' PAGE quantification and sequencing library pooling ''' <br> | |||
* I did PAGE quantification on the gel images above (300-700bp range) and pooled 75ng each library index 1-12 together. | |||
''' Pool 1: #1 - #12 ''' | |||
{| {{table}} border = 1 | |||
| align="center" style="background:#f0f0f0;"|'''Sample #''' | |||
| align="center" style="background:#f0f0f0;"|'''Index''' | |||
| align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''Yields in 45ul (ng)''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume for 75ng (ul)''' | |||
|- | |||
| #1||Index 1||9.34||420.25||8.03 | |||
|- | |||
| #2||Index 2||8.48||381.66||8.84 | |||
|- | |||
| #3||Index 3||8.10||364.43||9.26 | |||
|- | |||
| #4||Index 4||9.05||407.25||8.29 | |||
|- | |||
| #5||Index 5||8.09||363.98||9.27 | |||
|- | |||
| #6||Index 6||8.59||386.41||8.73 | |||
|- | |||
| #7||Index 7||10.35||465.54||7.25 | |||
|- | |||
| #8||Index 8||8.22||370.02||9.12 | |||
|- | |||
| #9||Index 9||9.30||418.65||8.06 | |||
|- | |||
| #10||Index 10||8.51||382.75||8.82 | |||
|- | |||
| #11||Index 11||10.79||485.45||6.95 | |||
|- | |||
| #12||Index 12||10.12||455.39||7.41 | |||
|} | |||
''' Pool 2: #13 - #24 ''' | |||
{| {{table}} border = 1 | |||
| align="center" style="background:#f0f0f0;"|'''Sample #''' | |||
| align="center" style="background:#f0f0f0;"|'''Index''' | |||
| align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''Yields in 45ul (ng)''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume for 75ng (ul)''' | |||
|- | |||
| #13||Index 1||10.65||479.03||7.05 | |||
|- | |||
| #14||Index 2||13.01||585.60||5.76 | |||
|- | |||
| #15||Index 3||11.26||506.66||6.66 | |||
|- | |||
| #16||Index 4||10.77||484.48||6.97 | |||
|- | |||
| #17||Index 5||7.14||321.12||10.51 | |||
|- | |||
| #18||Index 6||10.48||471.75||7.15 | |||
|- | |||
| #19||Index 7||10.66||479.57||7.04 | |||
|- | |||
| #20||Index 8||8.76||394.04||8.57 | |||
|- | |||
| #21||Index 9||12.23||550.38||6.13 | |||
|- | |||
| #22||Index 10||11.96||538.07||6.27 | |||
|- | |||
| #23||Index 11||8.76||394.36||8.56 | |||
|- | |||
| #24||Index 12||11.12||500.41||6.74 | |||
|}<br> | |||
''' 2013_05_25 '''<br> | |||
''' PAGE size-selection ''' | |||
* Each pool has ~900ng of 300-700bp fragments | |||
* Each pool purified in 1 of 6% 5-well TBE gel | |||
[[File:ZhangLab_2 2013-05-25 18hr 06min_ss-pool1.jpg| 300px]] [[File:ZhangLab_2 2013-05-25 18hr 13min_ss-pool2.jpg| 300px]] | |||
I loaded too much 100bp DNA ladder (500ng, 50ng/ul). The bands are too close, so difficult to locate 700bp band. | |||
Next time may load 250ng and run a little longer for a better resolution. | |||
* Resuspend each pool with total 40ul H2O | |||
<br> | |||
''' PAGE verification of sequencing libraries ''' | |||
* Loaded 1.5ul of each library pool in 6% TBE gel | |||
[[File:ZhangLab_2 2013-05-27 13hr 04min_EC-PGP1-PAGEverify.jpg| 150px]] | |||
P1 = EC-PGP1-Dev7-1-12-May23-2013 | |||
P2 = EC-PGP1-Dev7-13-24-May23-2013 | |||
*** This time, I cut the gel lower than expected 700bp bands (~600bp)!! | |||
''' Library ID, Pool1: EC-PGP1-Dev7-1-12-May23-2013 : ''' conc. by Qubit dsDNA HS (used 2ul for assay) = 3.51 ng/ul or '''10.79 nM''' <br> | |||
''' Library ID, Pool2: EC-PGP1-Dev7-13-24-May23-2013 :''' conc. by Qubit dsDNA HS (used 2ul for assay) = 3.56 ng/ul or '''10.95 nM''' <br> | |||
* MW of 500bp (average of 300-700bp) = 500bp * 650Da/bp + 159Da ~ 325,159Da or g/mole | |||
* Alan did qPCR to quantify the concentration of the libraries again | |||
'''GAII run: HL153 (SE)''' | |||
* EC-PGP1-Dev7-1-12-May23-2013: Lane 3 | |||
* EC-PGP1-Dev7-13-24-May23-2013: Lane 4 |
Latest revision as of 01:04, 30 May 2013
Adaptor ligation of CoRE fragmented DNA (Eric's May 23, 2013 experiment)[edit]
- Got 24 samples from Eric for ligation using NEBNext adaptor.
- MDA and CoRE fragmentation were performed by Eric and documented on Eric's note on May23, 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 17-24 --> 24-17.
- All procedures of adaptor ligation and amplification were the same as my experiment on May22, 2013
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 diluted a new batch of adaptor by diluting 15uM adaptor to 1.5uM (mix 20ul of 15uM NEBNext adaptor with 180ul H2O) and aliquoted 25ul into 8-striptubes and saved at -20C after used.
Ligation reaction mix
Ligation reaction mix | 1x rxn | 24x rxn mix |
2X KAPA Rapid Ligation Buffer | 25.00 | 600.00 |
KAPA Rapid T4 DNA Ligase | 2.00 | 48.00 |
H2O | 15.33 | 367.92 |
Total | 42.33 | 1,015.92 |
- 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 | 25 rxn mix |
adapter ligated DNA | 20.00 | 0.00 |
PCR_F (NEB, 10uM) | 1.00 | 25.00 |
NEBNext.IndX (NEB, 10uM) | 1.00 | 0.00 |
2X KAPA SYBR MM | 25.00 | 625.00 |
H2O | 3.00 | 75.00 |
Total volume | 50.00 | 725.00 |
- Aliquot 29ul, add 1ul of NEBNext Indx, 20ul of adapter ligated DNA
Samples | Indx | Samples | Indx |
#1 | NEBNext Index1 | #13 | NEBNext Index1 |
#2 | NEBNext Index2 | #14 | NEBNext Index2 |
#3 | NEBNext Index3 | #15 | NEBNext Index3 |
#4 | NEBNext Index4 | #16 | NEBNext Index4 |
#5 | NEBNext Index5 | #17 | NEBNext Index5 |
#6 | NEBNext Index6 | #18 | NEBNext Index6 |
#7 | NEBNext Index7 | #19 | NEBNext Index7 |
#8 | NEBNext Index8 | #20 | NEBNext Index8 |
#9 | NEBNext Index9 | #21 | NEBNext Index9 |
#10 | NEBNext Index10 | #22 | NEBNext Index10 |
#11 | NEBNext Index11 | #23 | NEBNext Index11 |
#12 | NEBNext Index12 | #24 | NEBNext Index12 |
Program (Eppendorf Realplex) 98C - 30s, (98C - 10s, 65C - 30s, 72C - 45s)x15, 72C - 2min, hold at 15C I stopped the reaction at 11 cycles
I plotted qPCR curves separately according to the gel loading order because I can't clearly observe amplification curve of each sample by plotting all 24 samples together. File:2013-05-24 Eric.qPCR 1-8.png File:2013-05-24 Eric.qPCR 9-16.png File:2013-05-24 Eric.qPCR 17-24.png
- 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 2013-05-24 15hr 41min 1-8.jpg File:ZhangLab 2 2013-05-24 15hr 43min 9-16.jpg File:ZhangLab 2 2013-05-24 15hr 46min 17-24 e.jpg
- All samples worked as expected since Eric did qPCR to verify the yield of MDA.
- The average size of libraries looked smaller than the ones on May22
- This time, I reduced the number of cycle from 12 cycles to 11 cycles. Next time with the same amount of input, I may reduce to 9 or 10 cycles to reduce bias from amplification.
PAGE quantification and sequencing library pooling
- I did PAGE quantification on the gel images above (300-700bp range) and pooled 75ng each library index 1-12 together.
Pool 1: #1 - #12
Sample # | Index | Conc. (ng/ul) | Yields in 45ul (ng) | Volume for 75ng (ul) |
#1 | Index 1 | 9.34 | 420.25 | 8.03 |
#2 | Index 2 | 8.48 | 381.66 | 8.84 |
#3 | Index 3 | 8.10 | 364.43 | 9.26 |
#4 | Index 4 | 9.05 | 407.25 | 8.29 |
#5 | Index 5 | 8.09 | 363.98 | 9.27 |
#6 | Index 6 | 8.59 | 386.41 | 8.73 |
#7 | Index 7 | 10.35 | 465.54 | 7.25 |
#8 | Index 8 | 8.22 | 370.02 | 9.12 |
#9 | Index 9 | 9.30 | 418.65 | 8.06 |
#10 | Index 10 | 8.51 | 382.75 | 8.82 |
#11 | Index 11 | 10.79 | 485.45 | 6.95 |
#12 | Index 12 | 10.12 | 455.39 | 7.41 |
Pool 2: #13 - #24
Sample # | Index | Conc. (ng/ul) | Yields in 45ul (ng) | Volume for 75ng (ul) |
#13 | Index 1 | 10.65 | 479.03 | 7.05 |
#14 | Index 2 | 13.01 | 585.60 | 5.76 |
#15 | Index 3 | 11.26 | 506.66 | 6.66 |
#16 | Index 4 | 10.77 | 484.48 | 6.97 |
#17 | Index 5 | 7.14 | 321.12 | 10.51 |
#18 | Index 6 | 10.48 | 471.75 | 7.15 |
#19 | Index 7 | 10.66 | 479.57 | 7.04 |
#20 | Index 8 | 8.76 | 394.04 | 8.57 |
#21 | Index 9 | 12.23 | 550.38 | 6.13 |
#22 | Index 10 | 11.96 | 538.07 | 6.27 |
#23 | Index 11 | 8.76 | 394.36 | 8.56 |
#24 | Index 12 | 11.12 | 500.41 | 6.74 |
2013_05_25
PAGE size-selection
- Each pool has ~900ng of 300-700bp fragments
- Each pool purified in 1 of 6% 5-well TBE gel
File:ZhangLab 2 2013-05-25 18hr 06min ss-pool1.jpg File:ZhangLab 2 2013-05-25 18hr 13min ss-pool2.jpg I loaded too much 100bp DNA ladder (500ng, 50ng/ul). The bands are too close, so difficult to locate 700bp band. Next time may load 250ng and run a little longer for a better resolution.
- Resuspend each pool with total 40ul H2O
PAGE verification of sequencing libraries
- Loaded 1.5ul of each library pool in 6% TBE gel
File:ZhangLab 2 2013-05-27 13hr 04min EC-PGP1-PAGEverify.jpg P1 = EC-PGP1-Dev7-1-12-May23-2013 P2 = EC-PGP1-Dev7-13-24-May23-2013 *** This time, I cut the gel lower than expected 700bp bands (~600bp)!!
Library ID, Pool1: EC-PGP1-Dev7-1-12-May23-2013 : conc. by Qubit dsDNA HS (used 2ul for assay) = 3.51 ng/ul or 10.79 nM
Library ID, Pool2: EC-PGP1-Dev7-13-24-May23-2013 : conc. by Qubit dsDNA HS (used 2ul for assay) = 3.56 ng/ul or 10.95 nM
- MW of 500bp (average of 300-700bp) = 500bp * 650Da/bp + 159Da ~ 325,159Da or g/mole
- Alan did qPCR to quantify the concentration of the libraries again
GAII run: HL153 (SE)
- EC-PGP1-Dev7-1-12-May23-2013: Lane 3
- EC-PGP1-Dev7-13-24-May23-2013: Lane 4