User Manual. Covaris DNA Shearing Verification Kit (PN ) INTRODUCTION KIT CONTENTS. STORAGE - 1 year at 4 C. WORKFLOW REVISION HISTORY

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1 Cvaris DNA Shearing Verificatin Kit (PN ) REVISION HISTORY PART NUMBER REVISION DATE DESCRIPTION OF CHANGE INTRODUCTION D 09/17 Remve bslete hlders, incrprate new instruments and cnsumables This kit allws users t rutinely verify the perfrmance f their Cvaris Fcused-ultrasnicatr. The kit may be used fr peridic assurance f perfrmance, r emplyed in trubleshting when applicatins perfrm differently than expected. The kit cntains a Reference Sample f genmic DNA, pre-fragmented, and a vlume f un-fragmented Test genmic DNA sufficient fr five perfrmance tests. Simply shear the Test DNA with yur Cvaris instrument and cmpare the results t the Reference, using the Agilent Bianalyzer 2100 (r equivalent). KIT CONTENTS The kit cntains sufficient material t perfrm up t 5 verificatin tests. This kit includes: - Reference Sample (Blue Cap): 40 µl f pre-fragmented DNA with an average fragment size distributin between 150 and 250 bp. - Test Sample (Red Cap): Tw tubes each cntaining 1100 µl f genmic DNA. SDS infrmatin is available at: STORAGE - 1 year at 4 C. WORKFLOW - Lad the recmmended vlume fr a given micrtube with Test Sample int 3 separate tubes. - Prcess these three samples fllwing instrument settings given in Table 1. Fr E- and LE-Series instruments, please psitin the tubes fllwing Table 2. The Reference Sample is already fragmented and des nt need t be further prcessed. - Analyze the fragment size distributin f bth Reference and Prcessed Test samples n the same chip. - Cmpare fragment size distributins t verify that yur Cvaris Fcused-ultrasnicatr is perfrming crrectly. Part number: Rev D Page 1 f 5

2 INSTRUMENT PARAMETERS / SETTINGS This kit is cmpatible with all micrtubes and assciated hlders / racks. Please fllw the settings carefully fr yur Cvaris Fcused-ultrasnicatr and the Cvaris micrtube that yu re using. Please be careful t lad the crrect vlume f sample, t use the matching rack / hlder and the intensifier, if applicable. Instrument micrtube Hlder / Rack / Insert M- M220 micrtube-50 Screw-Cap # Series # micrtube Snap-Cap # # micrtube-500 Screw- # # ME220 micrtube-15 Screw-Cap # (PN520145) 8 micrtube-15 Strip V2 # (PN / ) # micrtube-50 Screw-Cap # micrtube-50 Strip V2 # (PN / ) # micrtube-130 Screw- # Cap (PN520216) 8 micrtube-130 Strip V2 # (PN / ) # micrtube Pre-slit Snap- # Cap micrtube Crimp-Cap # micrtube Strip V1 # S-Series S2 micrtube Snap-Cap S220 micrtube-15 Screw-Cap (PN500145) micrtube-50 Screw-Cap micrtube Snap-Cap r Crimp-Cap micrtube-500 Screw- E-Series E220 / E220e micrtube-15 Screw-Cap (PN520145) Part number: Rev D Page 2 f 5 Temp Water Level Sample Vlume PIP / Intensity Duty Cycle/ Factr Time Cycles per Burst 20 C N/A 50 µl 75 W 10 % 195 s C N/A 130 µl 50 W 20 % 150 s C N/A 500 µl 75 W 20 % 210 s C µl 50 W 30 % 70 s C µl 50 W 30 % 70 s C µl 75 W 25 % 90 s C µl 50 W 30 % 125 s C µl 70 W 20 % 140 s C µl 70 W 20 % 130 s C µl 70 W 20 % 130 s C µl 70 W 20 % 140 s C µl 70 W 20 % 130 s 1000 # C µl I = 5 10 % 180 s 200 # C µl 18 W 20 % 120 s 50 # C µl 75 W 25 % 95 s 1000 # C µl 175 W 10 % 180 s 200 # C µl 175 W 20 % 180 s 200 # / # C µl 18 W 20 % 120 s 50

3 8 micrtube-15 Strip V2 # / 20 C 6 15 µl 18 W 20 % 120 s 50 (PN / ) # micrtube-50 Screw-Cap # / 7 C 6 55 µl 75 W 20 % 95 s 1000 # micrtube-50 Strip V2 # / 7 C µl 75 W 15 % 155 s 500 (PN / ) # micrtube-50 Plate N/A 7 C 0 55 µl 100 W 30 % 90 s 1000 (PN / ) * micrtube Snap-Cap # / 7 C µl 175 W 10 % 180 s 200 # micrtube Crimp-Cap # / 7 C µl 175 W 10 % 180 s 200 # micrtube Strip V1 # / 7 C µl 175 W 10 % 180 s 200 # micrtube Plate N/A 7 C µl 175 W 10 % 180 s 200 (PN / ) micrtube-500 Screw- # / 7 C µl 175 W 20 % 180 s 200 # L-Series LE220 8 micrtube-15 Strip V2 # C 4 15 µl 180 W 30 % 120 s 50 and LE220- (PN / ) ** 8 micrtube-50 Strip V2 # C µl 450 W 20 % 160 s 1000 plus (PN / ) 96 micrtube-50 Plate N/A 7 C µl 450 W 20 % 200 s 1000 (PN / ) *** micrtube Crimp-Cap # C µl 450 W 30 % 175 s micrtube Strip V1 # C µl 450 W 30 % 175 s micrtube Plate (PN / ) NA 7 C µl 450 W 30 % 190 s 200 Please nte while using the E220 and E220evlutin, the intensifier (PN500141) must remain in place fr all micrtubes with the exceptin f the micrtube-15. *Y-dithering functin (0.5mm Y-dither at 10mm/s) required. **Y-dithering functin (5mm Y-dither at 20mm/s) required. * X and Y-dithering functin (0.5mm X-dither & 0.5mm Y-dither at 10mm/s) required. These functins are nly available n SnLab versin 7.3 and up. Please refer t the DNA Shearing Quick Guide fr detailed instructins. Table 1 Cvaris Instrument DNA Shearing Settings Psitin f Sample #1 Psitin f Sample #2 Psitin f Sample #3 24 well rack A1 B3 D6 96 well rack A1 D6 H12 Table 2 Test samples psitin in an E r LE-Series Cvaris instrument Part number: Rev D Page 3 f 5

4 INTERPRETATION Fr analysis, emply the available analysis device (Agilent Bianalyzer 2100, Caliper LabChip, Agilent 2200 TapeStatin, Bi-Rad Experin, Agarse gel, etc.). It is imprtant t run bth the Reference and Prcessed Test Samples n the same chip r gel t nrmalize the results frm analytical assay variatins. Fr each sample, determine the peak size f the fragment distributin. Fr the three Prcessed Test Samples, calculate the average and the Cefficient f Variatin. Cmpare the peak size f the Reference and Prcessed Test Samples using Table 3. Average f Prcessed Test Samples within +/- 15% f Reference Sample Cvaris system OK Cefficient f Variatin f Prcessed Samples < 10% Cefficient f Variatin f Prcessed Samples > 10% Reference Sample in the bp Cvaris system OK range Reference Sample ut f the bp Prblem with fragment size range distributin analysis Table 3 Cvaris Perfrmance Verificatin Kit interpretatin Cvaris Cntact: Applicatinsupprt@cvaris.cm DETAILED INSTRUCTIONS FOR USING THE AGILENT BIOANALYZER 2100 Average f Prcessed Test Samples mre than 15% different frm Reference Sample - Lad bth Reference and Prcessed Test Samples n an Agilent Chip fllwing the manufacturer s instructins. 12k, 7.5k and 1k Chip: lad 1 µl f bth the Reference and Prcessed Test DNA Samples. High Sensitivity: dilute bth Reference and Prcessed Test DNA Samples 1:10 and lad 1 µl n the Chip. - Analyze the results. Open the Analysis tab n the right f the 2100 Expert Sftware by clicking n the dts and sliding yur muse n the left. In the Glbal tab, g in the Smear Analysis sectin and click n Perfrm Smear Analysis, then click n the dts fllwing Regins. Part number: Rev D Page 4 f 5

5 In the Regins windw, click n Add t add a new regin starting at 100 bp and finishing at 300 bp. Click OK. In the main windw, click n Regin Table at the bttm and nte the value fr % f Ttal and Average Size. Repeat the smear analysis fr the Reference Sample and each ne f the Prcessed Test Samples. A spike in the fragment distributin r bump in the baseline may happen in sme Agilent Bianalyzer runs and will significantly cmprmise the accuracy f the % f Ttal value. In this case, please re-run the samples n a new Chip. % f Ttal fr the Reference Sample shuld be > 50%. If < 50%, this indicates a prblem with the fragment size distributin analysis. Please check that the Bianalyzer is functining crrectly then repeat with a new chip. As an alternate t a peak size analysis, calculate the % f Ttal f the three Prcessed Test Samples, average these results, calculate the Cefficient f Variatin, and use Table 3 t determine the status f yur Cvaris Instrument. If the Cefficient f Variatin f the three Prcessed Test Samples is > 10% r if the average is > 15% different frm the Test Sample, cntact Cvaris at Applicatinsupprt@cvaris.cm The % f Ttal takes int accunt the area belw the upper and lwer marker, s the results are dependent n sample cncentratin and d nt reflect the actual area f the fragment distributin in the range f interest. It is therefre critical t lad the same vlume, and the same cncentratin f Reference and Prcessed Test Samples. Part number: Rev D Page 5 f 5