Quick Guide: DNA Shearing with S220 and E220

Quick Guide:
DNA Shearing with S220/E220 Focused-ultrasonicator
.
This Quick Guide provides DNA Shearing protocols when using microTUBE, microTUBE-50, microTUBE-15 or
miniTUBE and a Covaris S220 or E220 Focused-ultrasonicator.
130 µl sample volume- from 150 to 1,500 bp
microTUBE AFA Fiber Snap-Cap
microTUBE AFA Fiber Crimp-Cap
8 microTUBE AFA Fiber Strip
96 microTUBE Plate
Target BP (Peak)
150
200
300
400
500
800
1,000 1,500
Peak Incident Power (W)
Duty Factor
Cycles per Burst
Treatment Time (s)
175
10%
200
430
175
10%
200
180
140
10%
200
80
140
10%
200
55
105
5%
200
80
105
5%
200
50
105
5%
200
40
140
2%
200
15
Temperature (°C)
Water Level – S220
Water Level – E220
Sample volume (µl)
E220 - Intensifier (PN 500141)
7
12
6
130
Yes
7
12
6
130
Yes
7
12
6
130
Yes
7
12
6
130
Yes
7
12
6
130
Yes
7
12
6
130
Yes
7
12
6
130
Yes
7
12
6
130
Yes
50 µl sample volume - from 150 to 1,500 bp
microTUBE-50 AFA Fiber Screw-Cap
8 microTUBE-50 AFA Fiber Strip V2
96 microTUBE-50 AFA Fiber Plate
Target BP (Peak)
150
200
300
400
500
1,000 1,500
Peak Incident Power (W)
Duty Factor
Cycles per Burst
Treatment Time (s)
175
10%
200
340
175
10%
200
140
175
10%
200
58
175
10%
200
38
175
5%
200
55
175
2%
200
50
175
1%
200
20
Temperature (°C)
Water Level – S220
Water Level – E220:
Screw-Cap
Plate & Strip
Sample volume (µl)
E220 - Intensifier (PN 500141)
7
15
7
15
7
15
7
15
7
15
7
15
7
15
10
6
50
Yes
10
6
50
Yes
10
6
50
Yes
10
6
50
Yes
10
6
50
Yes
10
6
50
Yes
10
6
50
Yes
Part Number:
Date:
400103 Rev H
April, 2015
1|P a g e
15 µl sample volume - from 150 to 550 bp
microTUBE-15 AFA Beads Screw-Cap
8 microTUBE-15 AFA Beads Strip V2
Target BP (Peak)
150
200
250
350
550
Peak Incident Power (W)
Duty Factor
Cycles per Burst
Treatment Time (s)
18
20%
50
300
18
20%
50
120
18
20%
50
80
18
20%
50
45
18
20%
50
22
Temperature (°C)
Water Level – S220
Water Level – E220:
Screw-Cap
Strip
Sample volume (µl)
E220 - Intensifier (PN 500141)
20
15
20
15
20
15
20
15
20
15
10
6
15
No
10
6
15
No
10
6
15
No
10
6
15
No
10
6
15
No
To ensure reproducible DNA shearing, it is required to centrifuge samples before processing. Please
see Appendix A for instructions.
Please note that microTUBE-15 requires removal of the Intensifier (PN 500141) from the E220
focused-ultrasonicator. Please see Appendix B for instructions.
200 µl sample - 2,000; 3,000 and 5,000 bp
miniTUBE Clear, Blue or Red
Target BP (Peak)
2,000
3,000
5,000
miniTUBE
Clear
Blue
Red
Peak Incident Power (W)
Duty Factor
Cycles per Burst
Treatment Time (s)
3
20%
1,000
900
3
20%
1,000
600
25
20%
1,000
600
Temperature (°C)
Water Level – S220
Water Level – E220
Sample Volume (µl)
E220 - Intensifier (PN 500141)
7
15
11
200
No
20
15
11
200
No
20
15
11
200
No
Please note that miniTUBE requires removal of the Intensifier (PN 500141) from the E220 focusedultrasonicator. Please see Appendix B for instructions.
To fragment DNA to size larger than 5 kb, Covaris offers the g-TUBE: a single-use device that shears genomic
DNA into selected fragments sizes ranging from 6 kb to 20 kb. The only equipment needed is a compatible
bench-top centrifuge.
Part Number:
Date:
400103 Rev H
April, 2015
2|P a g e
Values mentioned in this Quick Guide are nominal values. The tolerances are as follow:
-
Temperature +/-2°C
Sample volume +/- 5µl (from 15 to 20 µl, +/- 1µl for the microTUBE-15)
Water Level +/- 1
Sample preparation guidelines
-
DNA input: from 100 ng to 5 µg purified DNA (1 µg for the microTUBE-15)
Buffer: Tris EDTA, pH 8.0
DNA quality: Genomic DNA (> 10 kb). For lower quality DNA, Covaris recommends setting up a time dose
response experiment for determining appropriate treatment times.
Recommended settings are subject to change without notice.
See following link http://www.covarisinc.com/wp-content/uploads/pn_400103.pdf for updates to this
document.
Part Number:
Date:
400103 Rev H
April, 2015
3|P a g e
Supplies
Covaris AFA tubes
microTUBE
microTUBE AFA Fiber Snap‐Cap (25)
microTUBE AFA Fiber Crimp‐Cap (25)
96 microTUBE Plate (for E220)
8 microTUBE Strip (12) (for E220)
microTUBE-50
microTUBE-50 AFA Fiber Screw‐Cap (25)
8 microTUBE-50 AFA Fiber Strip V2 (12) (for E220)
96 microTUBE-50 AFA Fiber Plate (for E220)
microTUBE-15
microTUBE-15 AFA Beads Screw‐Cap (25)
8 microTUBE-15 AFA Beads Strip V2 (12) (for E220)
miniTUBE
miniTUBE Clear (25)
miniTUBE Blue (25)
miniTUBE Red (25)
520045
520052
520078
520053
520166
520174
520168
500145
500159
520064
520065
520066
Preparation stations
microTUBE Prep Station Snap & Screw Cap
miniTUBE loading and unloading station
8 microTUBE Strip Prep Station
500330
500207
500327
Holders for S220
S-Series Holder microTUBE
Compatible with microTUBE Snap‐ and Crimp‐Cap
S-Series Holder microTUBE Screw-Cap
Compatible with microTUBE-50 AFA Fiber Screw-Cap
S-Series Holder microTUBE Screw-Cap LV
Compatible with microTUBE-15 AFA Beads Screw-Cap
S-Series Holder miniTUBE
Compatible with Clear, Blue and Red miniTUBE
500114
Rack 24 Place microTUBE Snap‐Cap
Rack 24 Place microTUBE Screw‐Cap
Rack 24 Place miniTUBE
Rack 12 Place 8 microTUBE Strip
Compatible only with PN 520053
Rack 12 Place 8 microTUBE Strip V2
Compatible with all Strips V2
Rack 96 Place microTUBE Crimp‐Cap
500111
500308
500205
500191
IE‐DNA (required for E220)
Fit microTUBEs in bench top micro centrifuges
g-TUBEs (10) and prep station
500141
520059
520079
Racks for E220
Intensifier
Centrifuge adapter
g-TUBE
Part Number:
Date:
400103 Rev H
April, 2015
500339
500427
500206
500444
500282
4|P a g e
Appendix A : microTUBE-15 centrifugation before DNA Shearing
1. Sample loading and centrifugation
Individual microTUBE-15
Carefully load microTUBE-15 into “S-Holder Screw-Cap LV”. If some of the sample splash up on the wall of the
microTUBE while snapping the microTUBE-15 in the Holder, then repeat the centrifugation step. All the liquid should be
at the bottom of the microTUBE-15 before to start the AFA treatment.
Rack of microTUBE-15 (E220)
Load and centrifuge microTUBE-15 as described above before placing the tubes in the Rack.
Rack of 8 microTUBE-15 Strip V2 (E220)
Place the 8 microTUBE-15 Strip V2 in the rack and load sample. Then centrifuge the rack at 300 RCF for 1 minute in a
swinging bucket centrifuge.
2. Sample processing
Use settings provided in page 2
3. Sample recovery
Part Number:
Date:
400103 Rev H
April, 2015
5|P a g e
Appendix B : Removing or Installing the Intensifier (Covaris PN 500141) from an E System
The 500141 Intensifier is a small inverted stainless steel cone centered over the E Series transducer by four stainless wires. The
wires are held by in a black plastic ring pressed into the transducer well.
If an AFA protocol requires “no intensifier”, please remove the Intensifier, using the following steps:
1.
2.
3.
Empty the water bath. Start the E System and start the SonoLAB software.
Wait for the homing sequence to complete (the transducer will be lowered with the rack holder at it home position,
allowing easy access to the Intensifier).
Grasp opposite sides of plastic ring and gently pull the entire assembly out of the transducer well. Do not pull on the steel
cone or the wires. The ring is a friction fit in the well – no hardware is used to hold it in place.
The 500141 Intensifier (left) shown installed in the E System transducer well and (right) removed.
Note the “UP” marking at the center of the Intensifier.
If a protocol requires the Intensifier to be present, simply reverse this process:
1. Align the black plastic ring with the perimeter of the transducer well. Note that the flat side of the center cone (marked UP)
should be facing up (away from the transducer).
2. Gently press each section of the ring into the well until the ring is seated uniformly in contact with the transducer, with
approximately 2 mm of the ring evenly exposed above the transducer assembly. Do not press on the cone or wires. The
rotation of the ring relative to the transducer assembly is not important.
3. Refill the tank. Degas and chill the water before proceeding.
Technical Assistance


By telephone (+1 781 932 3959) during the hours of 9:00am to 5:00pm, Monday through Friday, United States Eastern
Standard Time (EST) or Greenwich Mean Time (GMT) minus 05:00 hours
By e-mail at [email protected]
Part Number:
Date:
400103 Rev H
April, 2015
6|P a g e