Sam:LabNotes/Human/2009-4-25: Difference between revisions
Jump to navigation
Jump to search
>Sam Chiang |
>Sam Chiang |
||
(6 intermediate revisions by the same user not shown) | |||
Line 5: | Line 5: | ||
*Perform shut-gun sequencing in order to clarify the concern of TIPs existence in these samples. | *Perform shut-gun sequencing in order to clarify the concern of TIPs existence in these samples. | ||
*'''Bioruptor (Diagenode)''' | *'''An example of Bioruptor setting(Diagenode) '''[[File:DNA shearing using bioruptor.pdf]] | ||
==Procedures== | ==Procedures== | ||
Line 19: | Line 19: | ||
*Prepare other 4 tubes with 100uL H2O for balance in Bioruptor machine. | *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) | ||
**This program is designed to obtain 300~400bp fragments | |||
*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. | ||
*Switch (clockwise)the knob to "15min" and turn on the sonification. | *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. | *Perform three cycles of 15min sonification (45min in total). Take out the samples for brief vortexing and centrifuging during the interval. | ||
*Finished sample were stored in -20C. | *Finished sample were stored in -20C. | ||
==Results== | ==Results== | ||
[[File:sam050410-bioruptor sheared MDA amplicon.jpg| | [[File:sam050410-bioruptor sheared MDA amplicon.jpg|200px]] | ||
*Here is a Bioruptor poster which has very useful information on machine setting: | |||
**[[File:Bioruptor DNA shearing poster.pdf]] |
Latest revision as of 18:34, 12 October 2010
Bioruptor shearing on MDA amplicons (Bing Ren's lab)[edit]
Objectives[edit]
- 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.
- An example of Bioruptor setting(Diagenode) File:DNA shearing using bioruptor.pdf
Procedures[edit]
- 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)
- This program is designed to obtain 300~400bp fragments
- 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 (45min in total). Take out the samples for brief vortexing and centrifuging during the interval.
- Finished sample were stored in -20C.
Results[edit]
File:Sam050410-bioruptor sheared MDA amplicon.jpg
- Here is a Bioruptor poster which has very useful information on machine setting: