AlanFung:LabNotes/2015/2015-6-3: Difference between revisions

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>Alan6017518
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* Transfer sheared samples to non-stick 1.5mL tubes and label.<span style="background-color:#FFFF00">BR_X|Sheared|400bp</span>->Box(<span style="background-color:#0f0">Alan 2015/6/3 Dr. Rana WGBS</span>)
* Transfer sheared samples to non-stick 1.5mL tubes and label.<span style="background-color:#FFFF00">BR_X|Sheared|400bp</span>->Box(<span style="background-color:#0f0">Alan 2015/6/3 Dr. Rana WGBS</span>)
==End Repairing and A-Tailing==
==End Repairing and A-Tailing==
{| class="wikitable" class="wikitable" class="wikitable"
{| class="wikitable" class="wikitable" class="wikitable" class="wikitable"
|- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom"
|- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom"
| width="219" height="15" | Component
| width="219" height="15" | Component
| width="67" | Volume (uL)
| width="99" | Volume (uL)
| width="107" | Master Mix (uL)


|- style="font-size:12pt" align="center" valign="bottom"
|- style="font-size:12pt" align="center" valign="bottom"
| height="15" | Fragment avg 400bp, double-stranded DNA
| height="15" | Fragment avg 400bp, double-stranded DNA
| align="center" | 50
| align="center" | 50
| Do not add


|- style="font-size:12pt" align="center" valign="bottom"
|- style="font-size:12pt" align="center" valign="bottom"
| height="15" | End Repair & A-Tailing Buffer
| height="15" | End Repair & A-Tailing Buffer
| align="center" | 7
| align="center" | 7
| align="center" | 130.9


|- style="font-size:12pt" align="center" valign="bottom"
|- style="font-size:12pt" align="center" valign="bottom"
| height="15" | End Repair & A-Tailing Enzyme Mix
| height="15" | End Repair & A-Tailing Enzyme Mix
| align="center" | 3
| align="center" | 3
| align="center" | 56.1


|- style="font-size:12pt" align="center" valign="bottom"
|- style="font-size:12pt" align="center" valign="bottom"
| height="15" | Total Volume
| height="15" | Total Volume
| align="center" | 60
| align="center" | 60
| align="center" | 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.
{| class="wikitable" class="wikitable" class="wikitable" class="wikitable" class="wikitable"
|- style="background-color:#E5E5E5;font-size:12pt" align="center"
| width="219" rowspan="2" height="15" | Step
| width="99" rowspan="2" | Temp
| width="107" rowspan="2" | Time
|- style="background-color:#E5E5E5;font-size:12pt" align="center"
|- style="font-size:12pt" align="center"
| rowspan="4" height="15" | End Repair & A-Tailing
| rowspan="2" | 20 °C
| rowspan="2" | 30 min
|- style="font-size:12pt" align="center"
|- style="font-size:12pt" align="center"
| rowspan="2" | 65 °C
| rowspan="2" | 30 min
|- style="font-size:12pt" align="center"
|- style="font-size:12pt" align="center"
| rowspan="2" height="15" | HOLD
| rowspan="2" | 4 °C
| rowspan="2" | ∞
|- style="font-size:12pt" align="center"


|}
|}

Revision as of 23:33, 9 June 2015

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)
BR_1 21.7 18.43 91.57
BR_2 54 7.41 102.59
BR_3 57.3 6.98 103.02
BR_4 71.9 5.56 104.44
BR_5 39.5 10.13 99.87
BR_6 29.4 13.61 96.39
BR_7 33.7 11.87 98.13
BR_8 38.3 10.44 99.56
BR_9 32.1 12.46 97.54
BR_10 24 16.67 93.33
BR_11 40.3 9.93 100.07
BR_12 46.3 8.64 101.36
BR_13 27.8 14.39 95.61
BR_14 29.5 13.56 96.44
BR_15 35.2 11.36 98.64
BR_16 39 10.26 99.74
  • 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 & A-Tailing 20 °C 30 min
65 °C 30 min
HOLD 4 °C