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 113: Line 114:
* 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.
* 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>
''' 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.
* I did PAGE quantification on the gel images above (300-700bp range) and pooled 75ng each library index 1-12 together.
Line 187: Line 189:
  I loaded too much 100bp DNA ladder (500ng, 50ng/ul). The bands are too close, so difficult to locate 700bp band.  
  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.
  Next time may load 250ng and run a little longer for a better resolution.
* Resuspend each pool with total 40ul H2O
* Resuspend each pool with total 40ul H2O
<br>
''' PAGE verification of sequencing libraries '''
''' PAGE verification of sequencing libraries '''
* Loaded 1.5ul of each library pool in 6% TBE gel
* Loaded 1.5ul of each library pool in 6% TBE gel


''' Library ID, Pool1: EC-PGP1-Dev7-1-12-May23-2013 : ''' conc. by Qubit dsDNA HS (used 2ul for assay) = XXg/ul or XX nM <br>
[[File:ZhangLab_2 2013-05-27 13hr 04min_EC-PGP1-PAGEverify.jpg| 150px]]
''' Library ID, Pool2: EC-PGP1-Dev7-13-24-May23-2013 :''' conc. by Qubit dsDNA HS (used 2ul for assay) = XX ng/ul or XX nM <br>
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
* 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