AlanFung:LabNotes/2015/2015-6-3: Difference between revisions
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* Incubate at 20 °C for 15 min. | |||
* Proceed immediately to the next step. |
Revision as of 23:49, 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 | 20 °C | 30 min |
A-Tailing | 65 °C | 30 min |
HOLD | 12 °C | ∞ |
- Proceed immediately to the next step
Adapter Ligation
- Dilute adapter stocks to the appropriate concentration, as outlined in Table 1 on p. 4.
- 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 | 5 | 93.5 |
Ligation Buffer | 30 | 561 |
DNA Ligase | 10 | 187 |
Adapter stock | 5 | 93.5 |
Total volume | 110 | 935 |
- Incubate at 20 °C for 15 min.
- Proceed immediately to the next step.