DNA Shearing
Application Notes -Protocols - Presentations
Get the DNA size you want every time. (down to 50bp)100ul volumes and below.
One of the unique features of the Covaris AFA is the ability
to shear DNA. Double stranded DNA (unlike RNA) will shear when exposed
to the energy of AFA. This shearing is site independent and is a perfect
substrate for the production of DNA libraries.
The system is computer controlled and once the conditions
have been optimised for the size range you require it is highly repeatable.
In addition to this AFA allows for a large number of samples to be processed
in a short period of time with no washing between samples.
The instrument is an essential part of the emulsion PCR process used to prepare the beads for emulsion PCR.
The Covaris System is also used to shear DNA into 50 bp fragments for fragment library preparation.
Reproducability
Tunability
| Example data | |
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| click here for data | click here for data12 repeat sample |
Data from 100ul tubes The following was generated with "100ul" tubes with the G7 holder. This is the S2 holder with the Intensifier attached. Peal size from Bioanalyzer electropherogram, in triplicate. The nominal volume is 150ul, but this run was at 100ul TE buffer.
DNA Shearing Conditions
Base Pair size |
100 bp |
200 bp |
500 bp |
|
Duty cycle |
% |
20% |
20% |
5% |
Intensity |
0-10 |
5 |
5 |
3 |
Cycles Per Burst |
50-1000 |
200 |
200 |
200 |
frequency Sweep |
yes/no |
yes |
yes |
yes |
Z height |
mm |
6 |
6 |
6 |
Temperature |
degC |
6 |
6 |
6 |
Seconds (time) |
|
480 |
90 |
90 |
BP size may vary according to concentration volume etc |
|
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Results Range ~50-220bp @ 18 minutes Mid-point ~100 bp
*Covaris SOP acoustic shearing*
Starting material
Lambda dsDNA 48.5 KB, Tris EDTA buffer
Lanes 1,2,3 – increasing DNA concentration
(14-27ug/ml) all 6 minutes
lane 4
....DNA marker
lane 5 …..18minutes
lane 6 …..12 minutes
lane 7……6 minutes
The Broad Institute, have taken several steps to streamline and significantly reduce the amount of labor needed to conduct a sequencing using the 454 technology.
The Broad have focused on optimizing the sizing of DNA fragments, as sequencing is most effective when DNA fragments to be sequenced are in a narrow size range.
The Broad have now developed a new method to shear DNA more efficiently that generates the small fragments required with a higher yield than nebulization. This method uses Covaris AFA to shear the DNA in an enclosed environment.
Process Improvements for the Genome Sequencer 20
Advantages over current process:
Covaris AFA Shearing:
• Currently all DNA samples go through the Covaris process
• Non-contact shearing is performed in an enclosed tube
• Minimizes risk of sample cross contamination
• Less input DNA (3 micrograms; working towards 1microgram or less)
• Decrease shearing volume from 1.6 ml down to 0.5 ml (currently working on 0.1 ml)
• Scalable to 96 well format (E-series instrument) HPLC:
• Tight size distribution (200-600 bases)
• Automate separation of up to 96 libraries
Robotic Emulsion Breakage:
• Significantly increases walk-away time
• Eliminates learning curve associated with using filter syringes
• More consistent DNA bead recovery
• Reduce possibility of cross-contamination