AlanFung:LabNotes/2015/2015-6-3: Difference between revisions
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* Proceed immediately to the next step | * Proceed immediately to the next step | ||
==Adapter Ligation== | ==Adapter Ligation (2015-06-10)== | ||
* Assemble each Adapter Ligation reaction as follows: | * Assemble each Adapter Ligation reaction as follows: | ||
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Revision as of 00:17, 12 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) | 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 (2015-06-10)
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 (2015-06-10)
- 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.
- BR_X|Methylated|Adapter->Box(Alan 2015/6/3 Dr. Rana WGBS)
qPCR to check End Repair, A-tailing, and adapter ligation works (2015-06-11)
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
- All samples are amplified except #13.
- BR_X|No Meth|PCR->Box(Alan 2015/6/3 Dr. Rana WGBS)