Sam:LabNotes/Microbiome-new/2010-11-17
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MDA samples preparation for Covaris shearing-(single E.coli amplicons)[edit]
Background[edit]
- I want attempt to prepare more sheared E.coli SAGs (from short-vs-long ampification) for Illumina library construction.
- I didn't do any additional amplification on the short-amplified E.coli SAGs since the over-dose MDA reaction buffer may interfere the library construction.
Procedures[edit]
QUBIT quantification[edit]
File:Sam111710-qubit-new.jpg *IC = internal control of E.coli gDNA
- 4 pairs of samples(marked in red) were prepared for shearing:
- WGA102710-(2-1)- 3hr and 10hr
- WGA102710-(2-4)- 3hr and 10hr
- WGA110310-(2)- 4hr and 10hr
- WGA110310-(4)- 4hr and 10hr
Sample preparation[edit]
- Label the Covaris microtubes. Open the caps and leave tubes on 96-well rack.
- UV-treat the tubes and pipettes for 15min.
- Thaw the tRNA carrier (stored in -80C). Use the "2"-tRNA(96ng/uL)
Sample no. Sample name Conc.(ng/uL) Sample(uL) tRNA("2") H2O Total DNA(ng) --------------------------------------------------------------------------------------------------- HC01 WGA102710-(2-1)- 3hr 0.88 50.00 3.5 0.00 43.95ng HC02 WGA102710-(2-1)-10hr 729.07 0.68 - 49.00 500.00ng HC03 WGA102710-(2-4)- 3hr 0.98 50.00 3.5 0.00 48.95ng HC04 WGA102710-(2-4)-10hr 693.02 0.72 - 49.00 500.00ng HC05 WGA110310- (2) - 4hr 2.16 50.00 - 0.00 108.14ng HC06 WGA110310- (2) -10hr 661.63 0.75 - 49.00 500.00ng HC07 WGA110310- (4) - 4hr 3.09 50.00 - 0.00 154.65ng HC08 WGA110310- (4) -10hr 974.42 0.52 - 49.00 500.00ng *I didn't put any tRNA carrier for 4hr-amplified amplicons.
- 50uL sample suspension were carefully transferred into microtubes.
- Samples tubes were tapped on the rack and packed in a paper box with three disposable cooler.
- The package was shipped to the Sara through FedEx 11-17-'10 by Alice.
Follow up(11-18-'10)[edit]
- I noticed that I accidently did the wrong dilution for all of the 10hr-amplification samples. I load much less thant 500ng for shearing.
HC02 WGA102710-(2-1)-10hr HC04 WGA102710-(2-4)-10hr HC06 WGA110310- (2) -10hr HC08 WGA110310- (4) -10hr
- Since the concentrations are very high for these samples, I am planning to do the local Bioruptor shearing for these samples later.
Follow up(12-07-'10)[edit]
- Since Alice is going to shear her samples today. I prepared those four missed samples again in microtubes and sent to Sarah for shearing.
H2O DNA for 500ng HC02 WGA102710-(2-1)-10hr 49.0 0.68 uL HC04 WGA102710-(2-4)-10hr 49.0 0.72 uL HC06 WGA110310- (2) -10hr 49.0 0.75 uL HC08 WGA110310- (4) -10hr 49.0 0.52 uL
- Total volume 50uL. Shipped in RT (since all of them are in high DNA amount).