AlanFung:LabNotes/2015/2015-6-3

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Background

  • Noi made a batch of WGBS libraries on the whole blood DNA from Dr. Rana’s lab.
  • Dr. Rana has obtained some sequencing fund, and would like to get them sequenced.
  • If the libraries are good, can you tell Alan where to locate those libraries, so that we can send them to BioGem?
  • Noi's wiki [[1]]
  • Dinh found the gDNA samples from Dr. Rana in Noi's box.
  • Noi's wiki page shows that the library preparation failed and didn't have time to repeat experiment [[2]]
  • From the labels, Noi got about 1 ug of the samples and only used 200 ng, we should have 800ng for library prep.
  • I will repeat the library prep follow the exact protocol that you did with the tumor WGBS libraries?
  • Noi confirmed that the experiment failed for the first round of experiment and she did try a second time and still the result was not very good
  • She got very faint smear or barely see a smear in many samples by amplifying about 16-18 cycles, which is very unlikely for DNA input ~100ng
  • Noi doesn't know why this is the case but I will give a a try.

Covaris gDNA shearing (2015-06-09)

  • Called IGM to reserve the Covaris Shearing Machine.
  • I will shear 400ng of samples in case we need to repeat experiment again.
  • Transfer 400ng of samples and make up to 110ul with ddh2o
Sample Sample conc. (ng/ul) Volume for 400ng (ul) H2O (ul) Actual sample left
BR_1 21.7 18.43 91.57 Less than 10 ul
BR_2 54 7.41 102.59 OK
BR_3 57.3 6.98 103.02 OK
BR_4 71.9 5.56 104.44 OK
BR_5 39.5 10.13 99.87 Less than 10.13 but close
BR_6 29.4 13.61 96.39 Less than 10 ul
BR_7 33.7 11.87 98.13 Less than 10 ul
BR_8 38.3 10.44 99.56 Less than 10 ul
BR_9 32.1 12.46 97.54 Less than 10 ul
BR_10 24 16.67 93.33 Close to none remaining
BR_11 40.3 9.93 100.07 Less than 9.93 ul
BR_12 46.3 8.64 101.36 OK
BR_13 27.8 14.39 95.61 None Remaining
BR_14 29.5 13.56 96.44 Less than 10 ul
BR_15 35.2 11.36 98.64 Less than 10 ul
BR_16 39 10.26 99.74 Less than 10 ul
  • Transfer to Covaris tube at IGM
  • Perform shearing
Followed up the set up for Covaris shearing from previous experiment
Target BP 400
Tube Micro tube 
Duty Factor 10%
Peak Incident Power (w) 140
Cycle per Burst 200
Time (secs) 55
Volume 50ul
Temperature (c) 7 
Intensifier Yes
  • Transfer sheared samples to non-stick 1.5mL tubes and label.BR_X|Sheared|400bp->Box(Alan 2015/6/3 Dr. Rana WGBS)

End Repairing and A-Tailing

Component Volume (uL) Master Mix (uL)
Fragment avg 400bp, double-stranded DNA 50 Do not add
End Repair & A-Tailing Buffer 7 130.9
End Repair & A-Tailing Enzyme Mix 3 56.1
Total Volume 60 187
  • Premix buffer and enzyme mastermix in a tube, aliquot 10ul to each well in a strip tube.
  • Add 50ul samples to each well and mix by pipetting.
Step Temp Time
End Repair 20 °C 30 min
A-Tailing 65 °C 30 min
HOLD 12 °C
  • Proceed immediately to the next step

Adapter Ligation

  • Assemble each Adapter Ligation reaction as follows:
Component Volume (μl) MasterMix (μl)
End Repair & A-Tailing reaction product 60 Do not add
PCR-grade water 9 168.3
Ligation Buffer 30 561
DNA Ligase 10 187
Adapter stock 1 18.7
Total volume 110 935
  • Incubate at 20 °C for 15 min.
  • Proceed immediately to the next step.

Beads Purification

  • 0.8X beads purification and elute with 25ul ddh2o.

qPCR to check if the end repair, A-tailing, and adapter ligation work

Components 1rxn 8.5 rxn mix
Adapter ligated DNA 2 Do not Add
2.5uM PCR_F+PCR_R N2 IndX 2 Do not Add
2x KAPA SYBG MM 12.5 233.75
H2O 8.5 158.95
Total 25 212.5
98C 2min --> (98C 15sec -> 64C 20sec -> 72C 30sec) x 12 cycles --> 72C 3min -->  15C Hold

File:AlanPCR12015611.jpg

  • All samples are amplified except #13.