Sam:LabNotes/Human/2009-4-25: Difference between revisions
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>Sam Chiang |
>Sam Chiang |
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='''DNA shearing for constructing sequencing library'''= | ='''DNA shearing for constructing sequencing library'''= | ||
== | ==Objectives== | ||
*According to Illumina array analysis, the sample B3(03/31, L4N9-with Tre) and D5(04/01. N6-no Tre) were the best amplicons in single cell amplification. | *According to Illumina array analysis, the sample B3(03/31, L4N9-with Tre) and D5(04/01. N6-no Tre) were the best amplicons in single cell amplification. | ||
*Perform shut-gun sequencing in order to | *Perform shut-gun sequencing in order to clarify the concern of TIPs existence in these samples. | ||
*'''Bioruptor (Diagenode)''' for DNA shearing[http://www.diagenode.com/pages/bioruptor200.html] | *'''Bioruptor (Diagenode)''' for DNA shearing[http://www.diagenode.com/pages/bioruptor200.html] | ||
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*MDA amplicons were picked from samples (20uL/tube). | *MDA amplicons were picked from samples (20uL/tube). | ||
*Perform 85C 3 min inactivation using PCR thermocycler | *Perform 85C 3 min inactivation using PCR thermocycler | ||
* | *Measure DNA concentration using Nanodrop. | ||
**Transfer 5ug sample into 1.5-mL microcentrifuge tubes and add water into 100 uL. | **Transfer 5ug sample into 1.5-mL microcentrifuge tubes and add water into 100 uL. | ||
conc. Sample H2O (RT-PCR grade) | conc. Sample H2O (RT-PCR grade) | ||
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D5: 634 ng/uL 7.88 92.12 uL | D5: 634 ng/uL 7.88 92.12 uL | ||
*Prepare other 4 tubes with 100uL H2O for | *Prepare other 4 tubes with 100uL H2O for balance in Bioruptor machine. | ||
*Adjust the on and off interval (30 sec on with 30sec off) | *Adjust the on and off interval (30 sec on with 30sec off) | ||
*Place the sample tubes in tube holder and check the cooling water is circulating normally. | *Place the sample tubes in tube holder and check the cooling water is circulating normally. |
Revision as of 05:45, 28 April 2009
DNA shearing for constructing sequencing library
Objectives
- According to Illumina array analysis, the sample B3(03/31, L4N9-with Tre) and D5(04/01. N6-no Tre) were the best amplicons in single cell amplification.
- Perform shut-gun sequencing in order to clarify the concern of TIPs existence in these samples.
- Bioruptor (Diagenode) for DNA shearing[1]
Procedures
- MDA amplicons were picked from samples (20uL/tube).
- Perform 85C 3 min inactivation using PCR thermocycler
- Measure DNA concentration using Nanodrop.
- Transfer 5ug sample into 1.5-mL microcentrifuge tubes and add water into 100 uL.
conc. Sample H2O (RT-PCR grade) B3: 661 ng/uL 7.56 92.44 uL D5: 634 ng/uL 7.88 92.12 uL
- Prepare other 4 tubes with 100uL H2O for balance in Bioruptor machine.
- Adjust the on and off interval (30 sec on with 30sec off)
- Place the sample tubes in tube holder and check the cooling water is circulating normally.
- Switch (clockwise)the knob to "15min" and turn on the sonification.
- Perform three cycles of 15min sonification. Take out the samples for brief vortexing and centrifuging during the interval.
- Finished sample were stored in -20C.