MJ Mini Gradient Thermal Cycler Instruction Manual

Transcription

1 MJ Mii Gradiet Thermal Cycler Istructio Maual Catalog #PTC 1148

2 MJ Mii Gradiet Thermal Cycler Istructio Maual Catalog #PTC 1148

3 Legal Notices Copyright 2009, Bio-Rad Laboratories, Icorporated. All rights reserved. Reproductio i ay form, either prit or electroic, is prohibited without writte permissio of Bio-Rad Laboratories, Ic. Notice to Purchaser This base uit, Serial No., i combiatio with its immediately attached Bio-Rad sample block module(s), costitutes a thermal cycler whose purchase coveys a limited o-trasferable immuity from suit for the purchaser s ow iteral research ad developmet ad for use i huma i vitro diagostics ad all other applied fields uder oe or more of U.S. Patets 5,656,493, 5,333,675, 5,475,610 (claims 1, 44, 158, ad 167 oly), ad 6,703,236 (claims 1 7 oly), or correspodig claims i their o-u.s. couterparts, owed by Applera Corporatio. No right is coveyed expressly, by implicatio or by estoppel uder ay other patet claim, such as claims to apparatus, reagets, kits, or methods such as 5 uclease methods. Further iformatio o purchasig liceses may be obtaied by cotactig the Director of Licesig, Applied Biosystems, 850 Licol Cetre Drive, Foster City, Califoria 94404, USA. These products are covered by oe or more of the followig U.S. patets or their foreig couterparts owed by Eppedorf AG: U.S. Patet Nos. 6,767,512 ad 7,074,367. Effective February 1, Visit o the Web at I the U.S., call toll free at BIORAD ( )

4 Table of Cotets Explaatio of Symbols... 4 Safety Warigs ad Guidelies... 4 Electromagetic Iterferece ad FCC Warig... 5 Documetatio Covetios... 6 Chapter 1: Itroductio... 7 Chapter 2: Layout ad Specificatios Chapter 3: Istallatio Chapter 4: Operatio Chapter 5: Ruig Protocols Chapter 6: Creatig Programs Chapter 7: Editig Programs Chapter 8: Usig the Utilities Chapter 9: Maiteace Chapter 10: Troubleshootig Appedix A: Preistalled Protocols Idex I the U.S., call toll free at BIORAD ( ) Visit o the Web at 3

5 Explaatio of Symbols CAUTION: Risk of Dager! Wherever this symbol appears, always cosult ote i this maual for further iformatio before proceedig. This symbol idetifies compoets that pose a risk of persoal ijury or damage to the istrumet if improperly hadled. CAUTION: Risk of Electrical Shock! This symbol idetifies compoets that pose a risk of electrical shock if improperly hadled. CAUTION: Hot Surface! This symbol idetifies compoets that pose a risk of persoal ijury due to excessive heat if improperly hadled. Safety Warigs ad Guidelies Warig: Operatig the MJ Mii Peltier thermal cycler before readig this maual ca costitute a persoal ijury hazard. Oly qualified laboratory persoel traied i the safe use of electrical equipmet should operate this machie. Warig: Do ot ope or attempt to repair the MJ Mii cycler base, the power supply, the heat pump/sample block, or other accessory. Doig so will void your warraties ad ca put you at risk for electrical shock. Retur the MJ Mii cycler to the factory (US customers) or a authorized distributor (all other customers) if repairs are eeded. Warig: The sample blocks ca become hot eough durig the course of ormal operatio to cause burs or cause liquids to boil explosively. Wear safety goggles or other eye protectio at all times durig operatio. Warig: The MJ Mii cycler icorporate eutral fusig, which meas that live power may still be available iside the machies eve whe a fuse has blow or bee removed. Never ope the cycler base; you could receive a serious electrical shock. Opeig the base will also void your warraties. Safe Use Guidelies The MJ Mii thermal cycler is desiged to be safe to operate uder the followig coditios: Idoor use Altitude up to 2,000 m Temperature 5 C to 40 C Maximum relative humidity 80% for temperatures up to 31 C, decreasig liearly to 50% relative humidity at 40 C Mais supply voltage fluctuatios ot to exceed ±10% of the omial voltage Istallatio categories (overvoltage categories) II Pollutio degree 2 Electrical Supply, VAC, Hz, 400 W 4 Visit o the Web at I the U.S., call toll free at BIORAD ( )

6 Electromagetic Iterferece This device complies with Part 15 of the FCC Rules. Operatio is subject to the followig two coditios: (1) this device may ot cause harmful iterferece, ad (2) this device must accept ay iterferece received, icludig iterferece that may cause udesired operatio. This device has bee tested ad foud to comply with the EMC stadards for emissios ad susceptibility established by the Europea Uio at time of maufacture. This digital apparatus does ot exceed the Class A limits for radio oise emissios from digital apparatus set out i the Radio Iterferece Regulatios of the Caadia Departmet of Commuicatios. LE PRESENT APPAREIL NUMERIQUE N EMET PAS DE BRUITS RADIOELECTRIQUES DEPASSANT LES LIMITES APPLICABLES AUX APPAREILS NUMERIQUES DE CLASS A PRESCRITES DANS LE REGLEMENT SUR LE BROUILLAGE RADIOELECTRIQUE EDICTE PAR LE MINISTERE DES COMMUNICATIONS DU CANADA. FCC Warig Warig: Chages or modificatios to this uit ot expressly approved by the party resposible for compliace could void the user s authority to operate the equipmet. Note: This equipmet has bee tested ad foud to comply with the limits for a Class A digital device, pursuat to Part 15 of the FCC Rules. These limits are desiged to provide reasoable protectio agaist harmful iterferece whe the equipmet is operated i a commercial eviromet. This equipmet geerates, uses, ad ca radiate radiofrequecy eergy ad, if ot istalled ad used i accordace with the istructio maual, may cause harmful iterferece to radio commuicatios. Operatio of this equipmet i a residetial area is likely to cause harmful iterferece i which case the user will be required to correct the iterferece at his ow expese. Shielded cables must be used with this uit to esure compliace with the Class A FCC limits. Regardig FCC Compliace: Although this desig of istrumet has bee tested ad foud to comply with Part 15, Subpart B of the FCC Rules for a Class A digital device, please ote that this compliace is volutary, for the istrumet qualifies as a Exempted device uder 47 CFR (c), i regard to the cited FCC regulatios i effect at the time of maufacture. Warraty The MJ Mii thermal cycler ad associated accessories are covered by a stadard Bio-Rad warraty. Cotact your local Bio-Rad Laboratories office for the details of the warraty. I the U.S., call toll free at BIORAD ( ) Visit o the Web at 5

7 Documetatio Covetios Typographic Covetios Example <<PROCEED>> Italics Meaig The ames of keyboard keys are ecased i double agle brackets. Select Edit from the Mai Meu. This istrumet system is labeled, For Research Use Oly. Graphic Covetios The programmig screes displayed i the LCD widow ca display up to seve lies of text at oe time. Example New: Lid:100 C ABCD Vol: 20µL 1= 92.0 FOR 0:30 2= LOWER TEMP C: Termiology A programmig optio is termed selected whe the optio is highlighted. I this maual, the selected terms are outlied (see the example above). 6 Visit o the Web at I the U.S., call toll free at BIORAD ( )

8 1 Itroductio MJ Mii Thermal Cyclers... 8 Importat Safety Iformatio

9 MJ Mii Thermal Cyclers The MJ Mii thermal cycler features: Premium cyclig i a medium capacity cycler: the MJ Mii thermal cycler ca hold 48 x 0.2 ml tubes, 12 x 0.5 ml tubes or oe 48-well microplate Ability to program ad maitai a 16 C temperature gradiet, frot-to-back, alog the block for protocol optimizatio i a sigle ru Itegrated Hot Boet heated lid for oil-free thermal cyclig. The heated lid pressure may be maually adjusted to permit the seatig of differet vessels Ituitive software with a easy-to-read iterface for rapid iput of programs, which may cotai advaced protocol steps such as auto-time extesio, auto-temperature icremet, ad variable ramp rates. Choice of calculated sample temperature cotrol for highest speed ad accuracy or block cotrol for compatibility with protocols desiged for a variety of istrumet types. Space-savig desig for easy setup ad trasportatio. Istat Icubate feature for cotiuous-temperature icubatios. Customizable factory-istalled protocols. Importat Safety Iformatio Safe operatio of the MJ Mii thermal cycler begis with a complete uderstadig of how the machie works. Please read this etire maual before attemptig to operate the system. Do ot allow ayoe who has ot read this maual to operate this machie. Warig: The MJ Mii cycler ca geerate eough heat to iflict serious burs ad ca deliver strog electrical shocks if ot used accordig to the istructios i this maual. Please read the safety warigs ad guidelies at the frot of this maual, ad exercise all precautios outlied i them. Warig: Do ot block the MJ Mii cycler s air vets (see Figure 4 for locatios). Obstructig air vets ca lead to overheatig ad icreases the risk of electrical shock ad fire. 8 Visit o the Web at I the U.S., call toll free at BIORAD ( )

10 2 Layout ad Specificatios Frot View...10 Cotrol Pael...10 Back View...11 Bottom View...11 Specificatios

11 Layout of the MJ Mii thermal cycler Thumbwheel Heated lid closed Cotrol pael Figure 1. Frot view of the MJ Mii thermal cycler. Scree hot key LCD widow Block heatig/ coolig lights Pause key Selectio keys Proceed key Cacel key Figure 2. Cotrol pael of the MJ Mii thermal cycler. 10 Visit o the Web at I the U.S., call toll free at BIORAD ( )

12 Layout ad Specificatios Heated lid Power switch Jack for power cord USB ports Fuses Figure 3. Back view of the MJ Mii thermal cycler. Frot Air itake vets Back Air exhaust vets Figure 4. Bottom view of the MJ Mii thermal cycler. I the U.S., call toll free at BIORAD ( ) Visit o the Web at 11

13 Specificatios MJ Mii Thermal Cycler Thermal rage: Thermal accuracy: Thermal uiformity: Rampig speed: Sample capacity: Lie voltage: Frequecy: Power: Fuses: Displays: Ports: Memory: Weight: Size (W x D x H): C, but o more tha 30 C below ambiet temperature ±0.2 C of programmed 90 C, NIST-traceable ±0.4 C well-to-well withi 10 sec of arrival at 90 C average ramp rates are 1.5 C/sec, with a maximum rate of 2.5 C/sec 48 x 0.2 ml tubes, 12 x 0.5 ml tubes, or oe 48-wel microplate VAC rms (o adjustmet eeded amog voltages withi these rages) Hz sigle phase 400 W maximum Two 6.3 A, 250 V, 5 x 20 mm 64 x 128 backlit LCD Two USB ports for access by service persoel from Bio-Rad Laboratories 400 typical programs i 12 folders 4.1 kg 18 x 32 x 20 cm Specificatios Gradiet Temp gradiet accuracy: Thermal row uiformity: Calculator accuracy: Gradiet rage: Thermal Gradiet differetial rage: ± 0.2 C of programmed target at ed rows, 10 sec after the timer starts for the gradiet step, NIST traceable ±0.4 C, i row, well-to-well, withi 10 sec of reachig target temperature ±0.4 C of actual row temperature, NIST-traceable C 1 16 C 12 Visit o the Web at I the U.S., call toll free at BIORAD ( )

14 3 Istallatio Packig Checklist Settig Up the MJ Mii Istrumet Evirometal Requiremets Power Supply Requiremets Air Supply Requiremets

15 Packig Checklist After upackig the MJ Mii thermal cycler, check to see that you have received the followig: Oe MJ Mii thermal cycler Two spare fuses Oe power cord MJ Mii Gradiet Thermal Cycler Istructio Maual (this documet) If ay of these compoets is missig or damaged, cotact Bio-Rad Laboratories to obtai a replacemet. Please save the origial packig materials i case you eed to retur the istrumet for service. Settig Up the MJ Mii Istrumet The MJ Mii cycler requires miimal assembly: pluggig i the power cord. Isert the power cord plug ito its jack at the back of the machie (see fig. 3 for locatio of jack), the plug the cord ito a electrical outlet. Evirometal Requiremets Esure that the area where the thermal cycler is istalled meets the followig coditios, for reasos of safety ad performace: Noexplosive eviromet Normal air pressure (altitude below 2000 m) Ambiet temperature 5 31 C Relative humidity up to 80% Uobstructed access to air that is 31 C or cooler (see below) Protectio from excessive heat ad accidetal spills. (Do ot place the MJ Mii thermal cycler ear heat sources, such as radiators, ad protect it from the dager of havig water or other fluids splashed o it, which ca cause shortig i its electrical circuits.) Power Supply Requiremets The MJ Mii thermal cycler requires V, Hz, ad a grouded outlet. This cycler ca use curret i the specified rage without adjustmet, so there is o voltage-settig switch. Power cords for outlets other tha the US 120 V outlet may be purchased from computer stores, sice they are also used for most desktop computers ad priters ad meet iteratioal stadard IEC The power cord must be rated to carry at least 10 A at 125 V or 250 V, depedig o the voltage available i your atio. The quality of the power cord ca be further esured by makig certai it is iscribed with the trademark of UL, CSA, TUV, VDE, or aother atioal testig agecy. Note: Do ot cut the supplied 120 V power cord ad attach a differet coector. Use a oe-piece molded coector of the type specified above. 14 Visit o the Web at I the U.S., call toll free at BIORAD ( )

16 istallatio Air Supply Requiremets The MJ Mii thermal cycler requires a costat supply of air. Air is take i from vets at the frot of the istrumet is exhausted from vets at the back (see fig. 4). If the air supply is iadequate or too hot, the machie ca overheat, causig performace problems, software error messages (particularly HS Overheatig ad Slow Block Cyclig ), ad eve automatic shutdows. Esurig a Adequate Air Supply Do ot block the air itake vets. Positio the MJ Mii thermal cycler at least 10 cm from vertical surfaces ad other thermal cyclers (greater distaces may be required; see below). Do ot put loose papers uder or i frot of the machie; they ca be sucked ito the air vets ad cause problems. Do ot allow dust or debris to collect i the air itake vets. The air vets are particularly liable to collect dust ad debris, sometimes completely cloggig up. Check for dust ad debris every few moths, ad clea the itake vets as eeded. Remove light collectios of dust with a soft-bristle brush or damp cloth. Severe collectios of dust ad debris should be vacuumed out. Tur the machie off prior to cleaig or vacuumig air vets. Esurig That Air Is Cool Eough Do ot positio two or more thermal cyclers so that the hot exhaust air of oe blows directly ito the air itake vets of aother. Make sure the MJ Mii thermal cycler receives air that is 31 C or cooler by measurig the temperature of air eterig the machie through its air itake vets. Air itake vets are located at the frot of the machie (see fig. 4). Place the thermal cycler where you pla to use it, ad tur it o. Try to reproduce what will be typical operatig coditios for the machie i that locatio, particularly ay heat-producig factors (e.g., earby equipmet ruig, widow blids ope, lights o). Ru a typical protocol (e.g., BASIC) for 30 miutes to warm up the cycler, the measure the air temperature at the air itake vets. If more tha oe machie is ivolved, measure the air temperature for each. If the air itake temperature of ay machie is warmer tha 31 C, use table 1 to troubleshoot the problem. Some experimetatio may be required to determie the best solutio whe more tha oe cause is ivolved. After takig steps to solve the problem, verify that the temperature of the air eterig the air itake vets has bee lowered, usig the procedure outlied above. Table 1. Troubleshootig air supply problems Cause Air circulatio is poor. Ambiet air temperature is high. Machie is i warm part of room. Machies are crowded. Possible Remedies Provide more space aroud machie or adjust room vetilatio. Adjust air coditioig to lower ambiet air temperature. Move machie away from, or protect machie from, such heat sources as radiators, heaters, other equipmet, or bright sulight. arrage machies so that warm exhaust air does ot eter itake vets. I the U.S., call toll free at BIORAD ( ) Visit o the Web at 15

17 16 Visit o the Web at I the U.S., call toll free at BIORAD ( )

18 4 Operatio Turig the MJ Mii Cycler O Uderstadig the Mai Meu Usig the Cotrol Pael Opeig ad Closig a Sample Block Selectig the Correct Sample Vessel Sealig Sample Vessels Loadig Sample Vessels ito the Block Tube, Microplate, ad Sealig System Selectio Chart Safety Warig Regardig Use of 35 S Nucleotides

19 Turig the MJ Mii Cycler O Move the power switch to o (the O positio). The fa will tur o, ad the Coolig/Heatig lights o the keyboard will flash. The MJ Mii cycler will eter a self-test of the heat pumps. Note: If the sample block or the heat sik is ot at ambiet temperature (typically because the sample block was recetly i use), the machie will skip the self-test. If the self-test does ot detect ay problems, the Mai Meu is displayed. Alteratively, depressig the «Scree» butto will brig you to the mai scree. RUN VIEW NEW FILES EDIT TOOLS Block is idle The MJ Mii cycler is ow ready to execute programs. Uderstadig the Mai Meu The Mai Meu is the commo access poit to all programmig ad machie cofiguratio screes: RUN: Executes a program. NEW: Allows ew programs to be etered. EDIT: Allows modificatio of stored programs. VIEW: Accesses utilities that display a program s steps. FILES: Accesses file maagemet utilities. TOOLS: Accesses machie cofiguratio screes. Usig the Cotrol Pael The cotrol pael (see fig. 2) icludes operatio keys, status idicator lights for heatig ad coolig, a LCD widow for displayig programmig ad machie status text, ad a umeric keypad for eterig values ito programs. Operatio Keys Select keys (left, right, up, dow arrows): Move the cursor oe space or optio i the LCD widow. Proceed/Eter: Accepts a selected meu or scree optio; durig a protocol ru, advaces the program to its ext step. Cacel: Termiates a ruig protocol; durig program creatio or editig, cacels the last etry. Pause: Pauses a protocol durig executio. Istat: Iitiates a program that sets up the MJ Mii as a simple icubator. 18 Visit o the Web at I the U.S., call toll free at BIORAD ( )

20 operatio Scree: Switches betwee protocol status screes, the Mai Meu ad a time remaiig display i the LCD widow durig a protocol ru. I additio, this butto toggles betwee the Mai Meu ad the About scree for the istrumet, ad allows the users to alterately view the text ad graphical programmig screes. Status Idicator Lights Block Status lights: Idicate whether the sample block is heatig (red light is illumiated) or coolig (blue light is illumiated). Opeig ad Closig a Sample Block Grip the frot hadle of the Hot Boet heated lid ad pull upward firmly. The top lever will pop ope to reveal the etire thumbwheel. Cotiue pullig upward to ope the Hot Boet lid. The Hot Boet lid will tip backward, revealig the etire block. Cautio: Do ot pull o the thumbwheel to ope the uit. This ca damage the closig mechaism of the Hot Boet lid. To close the sample block, press dow o the top lever. The lever will close dow over the thumbwheel as the Hot Boet lid closes dow over the sample block. A click sigifies that the lid s latch has egaged. Selectig the Correct Sample Vessel The MJ Mii cycler accepts both 0.2 ml ad 0.5 ml tubes as well as 48-well microplates. Keep i mid that differeces i tube ad plate compositio ad wall thickess amog the may brads available ca affect reactio performace. Protocols may require some adjustmet to esure optimum performace whe usig a ew vessel type. Bio-Rad offers a full rage of tubes ad microplates maufactured to the specificatios of the sample block of MJ Mii cycler. See Table 2 for a complete list. 0.5 ml Tubes It is possible to load up to twelve 0.5 ml tubes i the MJ Mii cycler. Sice the width of the 0.5 ml tubes does ot allow them to be accommodated i adjacet wells, the tubes should be placed i a staggered fashio (i.e., i every other well i the block). The tubes should NOT be placed i the spaces betwee wells. Furthermore, placemet of tubes i the peripheral wells alog the edge of the cycler block should be avoided, as this may result i compromised amplificatio due to codesate formatio. For correct placemet of 0.5 ml tubes, see Figure 5. Whe usig 0.5 ml tubes, thi-walled tubes are recommeded; these are specifically desiged for thermal cyclig ad the higher-quality brads provide a good ad cosistet fit. If thick-walled 0.5 ml tubes are used, esure that they fit the wells sugly. (Sice these tubes were origially desiged for cetrifuges, some brads may ot fit tightly i thermal cycler wells.) Both thi ad thick-walled 0.5 ml tubes are available from Bio-Rad ad are desiged for precise block fit. Figure 5. Wells (3) i which 0.5 ml tubes should be placed I the U.S., call toll free at BIORAD ( ) Visit o the Web at 19

21 Thi-Walled Vs. Thick-Walled Tubes The thickess of sample tubes directly affects the speed of sample heatig ad thus the amout of time required for icubatios. Thick-walled tubes delay sample heatig, sice heat trasfers more slowly through the tubes walls. For the earliest types of thermal cyclers this delay mattered little. These machies rampig rates were so slow (below 1 C/sec) that there was plety of time for heat to trasfer through the tube wall to the sample, durig a give icubatio. Moder thermal cyclers have much faster rampig rates, so the faster heat trasfer provided by thiwalled tubes allows protocols to be sigificatly shorteed. Essetially, up to 30 secods ca be saved per cycle by usig thi-walled tubes, for a overall savigs of 15 miutes i a 30-cycle ru. 0.2 ml Tubes All types of thi-walled 0.2 ml tubes may be used. Bio-Rad sells high-quality 0.2 ml tubes i a umber of styles, icludig idividual tubes ad strips. Microplates A variety of polypropylee 48-well microplates ca be used i the MJ Mii cycler as log as they fit the wells sugly. I additio, the Multiplate lie of 96-well plates ad Microseal film seals ca be cut dow to fit i the sample block of this istrumet. Polypropylee microplates ad compatible Microseal film or strip caps for sealig are available from Bio-Rad Laboratories. Sealig Sample Vessels To avoid chagig the cocetratio of reactats, steps must be take to prevet the evaporatio of water from reactio mixtures durig thermal cyclig. Oly a layer of oil or wax will completely prevet evaporatio from the surface of the reactio fluid. However, a adequate degree of protectio ca be achieved by sealig vessels with caps, film, or adhesive seals the cyclig the samples usig the heated lid to prevet codesatio. Sealig with Oil or Wax Mieral oil, silicoe oil, paraffi wax, or Chill-out liquid wax may be used to seal samples. Use oly a small amout of oil or wax; 1-3 drops (15 50 µl) are usually sufficiet. (Iclude this volume i the total volume whe settig up a calculated-cotrol protocol; see Choosig a Temperature Cotrol Mode i chapter 5.) Use the same amout of oil or wax i all sample vessels to esure a uiform thermal profile. Most paraffi waxes solidify at room temperature. The wax ca the be pierced with a micropipette tip ad the samples draw off from below the wax. Silicoe oil ad mieral oil ca be poured off or aspirated from tubes if the samples are first froze ( 15 to 20 C). The samples are usually pure eough for aalysis without a extractio. Chill-out liquid wax (available from Bio-Rad Laboratories) is a easy-to-use alterative to oil. This purified paraffiic oil solidifies at 10 C ad is liquid at room temperature. By programmig a holdstep at low temperature, the wax ca be solidified at the ed of a ru. A pipette tip ca the be used to pierce the wax i the tubes ad remove the samples. The wax is available i a clear, optical-assay grade or dyed red to assist i moitorig its use. The red dye has o adverse effects o fluorescet gel aalysis of reactio products. 20 Visit o the Web at I the U.S., call toll free at BIORAD ( )

22 operatio Sealig with the Hot Boet Lid The Hot Boet heated ier lid maitais the air i the upper part of sample vessels at a higher temperature tha the reactio mixture. This prevets codesatio of evaporated water vapor oto the vessel walls ad lid, so that solutio cocetratios are uchaged by thermal cyclig. The Hot Boet lid also exerts pressure o the tops of vessels loaded ito the block, helpig to maitai a vapor-tight seal ad to firmly seat tubes or the plate i the block. Caps, film, adhesive seals, or mats must be used alog with the Hot Boet lid to prevet evaporative losses. Note: Whe tubes are cooled to below-ambiet temperatures, a rig of codesatio may form i tubes above the liquid level but below the top of the sample block. This is ot a cause for cocer sice it occurs oly at the fial cool-dow step, whe thermal cyclig is complete. Microseal A film offers a quick alterative to sealig microplates or arrays of tube strips. This film is specially desiged to seal tightly durig cyclig, yet release smoothly to miimize the risk of aerosol formatio ad cross-cotamiatio of samples. Microseal A film, desiged to cover a 96-well plate, is easily cut for use with fewer tha 96 samples. Microseal B adhesive seals feature a aggressive adhesive, effective from 20 C to 110 C, which allows secure sample storage or trasport before ad after cyclig. The clear polyester backig allows easy ispectio of sample wells. Microseal B clear, adhesive seals are ideal for thermal cyclig i all polypropylee ad polystyree microplates. Microseal B adhesive seals ca be easily cut for use with fewer tha 96 samples. Microseal F alumiized foil acts as a barrier agaist evaporatio from 20 C to 105 C. I additio to cold storage applicatios, it ca also be used for thermal cyclig sample volumes 25 µl. The foil is thi eough to pierce with a pipet tip for recovery of sample from idividual wells. Microseal F foil is easily cut for use with fewer tha 96 samples. Adjustig the Hot Boet Lid s Pressure The pressure exerted by the Hot Boet lid must be maually adjusted to fit the sample vessels beig used. Oce set, the Hot Boet lid ca be opeed ad closed repeatedly without readjustmet as log as either the tube or microplate type or the sealig method is chaged. Ay chage i vessel type or sealig method requires readjustmet of the Hot Boet lid. Follow these steps to adjust the pressure exerted by the ier lid: 1. Make sure the block s wells are clea. Eve tiy amouts of extraeous material ca decrease thermal coductace ad iterfere with the proper seatig of a microplate or tubes. 2. Ope the Hot Boet lid. Tur the thumbwheel all the way couterclockwise to completely raise the ier lid. 3. Load either a microplate or at least eight idividual tubes ito the sample block. The ier lid pivots aroud a cetral poit, so it is importat to distribute idividual tubes evely: load at least four tubes i the ceter of the block ad at least oe tube i each of the four corers of the block. If usig a sealig film or mat, apply it to the loaded microplate accordig to the maufacturer s directios. 4. Close the Hot Boet lid by pressig dow o the top lever. Tur the thumbwheel clockwise to lower the ier lid oto the loaded microplate/tubes. The thumbwheel turs easily at first sice the ier lid has ot yet come ito cotact with aythig. Stop turig the thumbwheel whe you feel icreased resistace, which idicates that the ier lid has touched the microplate/tubes. I the U.S., call toll free at BIORAD ( ) Visit o the Web at 21

23 5. For microplate sealig films or mats that require additioal pressure, tur the thumbwheel clockwise a extra half tur past the poit of iitial cotact to set a appropriate lid pressure. Cautio: Do ot tur the thumbwheel more tha three-quarters of a tur. This ca make it hard or impossible to close the lid ad puts excessive strai o the latch holdig the lid closed. A extra half to three-quarters of a tur esures the correct pressure for most types of reactio vessels. Some empirical testig may be required to determie the optimum pressure required for certai vessels. Oce this pressure has bee determied, the thumbwheel positio may be marked with a colored markig pe or piece of tape. Note: As a aid i gaugig how much the thumbwheel has bee tured, mark it at the quarter tur positios, or every sixth bump o the thumbwheel (there are 24 total bumps ). Loadig Sample Vessels ito the Block Whe usig a small umber of tubes, load at least oe empty tube i each corer of the block to esure that the Hot Boet lid exerts eve pressure o the sample tubes (see Adjustig the Hot Boet Lid s Pressure, above). To esure uiform heatig ad coolig of samples, sample vessels must be i complete cotact with the block. Adequate cotact is esured by always doig the followig: Esure that the block is clea before loadig samples (see chapter 9 for cleaig istructios). Firmly press idividual tubes or the microplate ito the block wells. Usig Oil to Thermally Couple Sample Vessels to the Block With two exceptios (see below), Bio-Rad does ot recommed usig oil to thermally couple sample vessels to the block, for the followig reasos: Calculated-cotrol protocols do ot ru accurately whe oil is used. Oil traps dirt, which iterferes with thermal cotact betwee vessels ad the block. Calculated-cotrol protocols do ot ru accurately whe oil is used. Oil traps dirt, which iterferes with thermal cotact betwee vessels ad the block. Cautio: If you use oil i the block, use oly mieral oil. Never use silicoe oil. It ca damage the sample block. Oe exceptio to this recommedatio ivolves the use of volatile radioactive 35S ucleotides. A small amout of oil i the block ca help prevet escape of these compouds. See page 24 for importat iformatio regardig safe use of these compouds i polypropylee tubes ad polypropylee ad polycarboate microplates. A secod exceptio ivolves the use of thick-wall 0.5 ml tubes. Certai brads of these tubes fit poorly i the block, i which case, oil may somewhat improve thermal cotact. Wheever possible, use high-quality thi-wall tubes iteded for thermal cyclig (see page 23 for a tube ad plate selectio chart). 22 Visit o the Web at I the U.S., call toll free at BIORAD ( )

24 operatio Tube, Microplate, ad Sealig System Selectio Chart The followig sample vessels ad sealig optios are recommeded for use with the MJ Mii thermal cycler. These items are available from Bio-Rad Laboratories. Key 4 Reactio vessel fits block/sealig optio fits reactio vessel without modificatio. # Reactio vessel/sealig optio ca be cut to fit. Table 2. Compatible sample vessels ad sealig optios. Thermal Cycler Reactio Vessels Sealig Optios for Oil-free Cyclig Microseal Microseal Microseal Chill-out Bio-Rad A Film B Seal F foil Strip caps wax MJ Mii Descriptio Catalog # MSA-5001 MSB-1001 MSF-1001 TCS-series CHO-series Multiplate uskirted MLP-series well microplates MLL-series Multiplate uskirted MLP-series well microplates Multiplate uskirted MLP well microplates 0.2 ml strip tubes TBS-series /strip & 12/strip TLS-series 0.2 ml idividual tubes TFI TBI-series 0.5 ml idividual TBI-series 3 tubes, w/ caps Reactio vessel fits block/sealig optio fits reactio vessel without modificatio. Reactio vessel/sealig optio ca be cut to fit. I the U.S., call toll free at BIORAD ( ) Visit o the Web at 23

25 Safety Warig Regardig Use Of 35 S Nucleotides Some researchers have experieced a problem with radioactive cotamiatio whe usig 35 S i thermal cyclers. This problem has occurred with all types of reactio vessels. The Problem Whe 35 S ucleotides are thermally cycled, a volatile chemical breakdow product forms, probably SO 2. This product ca escape the vessel ad cotamiate the sample block of a thermal cycler, ad possibly, the air i the laboratory. Cotamiatio has bee reported with microplates, 0.2 ml tubes, ad 0.5 ml tubes. Polycarboate Reactio Vessels The largest risk of cotamiatio occurs whe usig 96-well polycaboate microplates. Polycarboate is somewhat permeable both to water ad the 35 S breakdow product. This problem is exacerbated whe polycarboate plates are held at high temperatures for log periods of time, or whe the plates are sealed for oil-free thermal cyclig. Polypropylee Reactio Vessels Polypropylee tubes ad microplates are maufactured with very thi walls to ehace thermal trasfer. The thi walls are somewhat fragile ad ca craze or develop small cracks whe subject to mechaical stress. Udamaged thi polypropylee tubes may also be somewhat permeable to the 35 S breakdow product. Either way, there have bee reports of 35 S passig through the walls of 0.2 ml polypropylee tubes of several differet brads durig thermal cyclig. Cotamiatio problems are rarer with 0.5 ml polypropylee tubes, but istaces have bee reported. No data are yet available o radioactive cotamiatio with polypropylee microplates. The Solutio 1. Substitute the low-eergy beta emitter 33 P i cycle sequecig. 33 P ucleotides are ot subject to the same kid of chemical breakdow as 35 S ucleotides, ad they have ot bee associated with volatile breakdow products. 2. If 35 S must be used, three thigs will help cotrol cotamiatio: a oil overlay iside the tubes, mieral oil i the thermal cycler outside the tubes, ad use of thick-walled 0.5 ml tubes. Always ru 35 S thermal cyclig reactios i a fume hood, ad be aware that vessels may be cotamiated o the outside after thermal cyclig. Please be certai that you are usig the appropriate detectio methods ad cleaig procedures for this isotope. Cosult your radiatio safety officer for his or her recommedatios. If mild cleaig agets do ot remove radioactivity, harsher cleaers may be used occasioally ad carefully. Users have suggested the deterget PCC-54 (Pierce Chemical Co., Rockford, Illiois; Pierce Eurochemie B.V., Hollad), Micro Cleaig Solutio (Cole-Parmer, Niles, Illiois), ad Dow Bathroom Cleaer (available i supermarkets). Cautio: Harsh cleaig agets (such as those above) are corrosive ad must be thoroughly rised away withi a few miutes of applicatio. They ca eat away the surface fiish of the blocks. 24 Visit o the Web at I the U.S., call toll free at BIORAD ( )

26 5 Ruig Protocols Ruig a Protocol Readig the Rutime Scree Readig the Temperature Display Scree Readig the Protocol Completio Scree Maually Steppig Through a Protocol Pausig a Ruig Protocol Stoppig a Ruig Protocol Resumig a Protocol After a Power Outage Usig the Istat Icubatio Feature

27 Ruig a Protocol Ruig a protocol o the MJ Mii thermal cycler ivolves two steps: 1. Choosig a stored protocol to ru 2. Settig up the temperature cotrol method Either a custom-desiged protocol or oe of the factory-istalled residet protocols may be ru. See appedix A for descriptios of the residet protocols, which may be edited to fit your eeds (see chapter 7 for istructios o editig stored programs). All the factory-istalled protocols are stored i a sigle folder, called the <MAIN> folder, at the time of shippig. Choosig a Stored Protocol to Ru With the Mai Meu displayed, select Ru, the press «Proceed». Oe of two types of scree will be displayed, depedig o whether custom protocols have bee stored i the <MAIN> folder or i custom folders: If all protocols have bee stored i the <MAIN> folder: A scree listig the protocols will be displayed. Custom protocols are listed first, followed by the factoryistalled programs: Ru: <MAIN> PROGRAMS iprf1kb iprf8kb iprf15kb LONG-2 LONG-3 Use the Select keys to scroll through the listed protocols. Scroll past the last- or first-listed protocol to see the ext scree dow or up. Select the desired protocol, the press «Proceed». If custom protocols have bee stored i oe or more custom folders: A list of folders will appear o the left of the scree ad a list of programs residig i these folders will appear o the right. I the example below, sice the MAIN folder is highlighted, the list of programs that reside i the MAIN folder appears o the right. Ru: PROGRAMS iprf1kb <FOLDER1> iprf8kb <FOLDER2> iprf15kb LONG-2 LONG-3 Select the folder that cotais the protocol you wish to ru, the press the right «Select» key to toggle over to the list of protocols located i that folder. Use the Select keys to scroll through the listed protocols ad select the desired protocol, the press «Proceed». 26 Visit o the Web at I the U.S., call toll free at BIORAD ( )

28 Ruig Protocols I either istace, after you press «Proceed», a scree similar to the oe below will be displayed: Ru: iprf1kb? Sample Vol: 20µl RUN VIEW The top lie of the scree will idetify the selected protocol (iprf1kb i the example). The other lies o the scree will request iformatio eeded to set up the temperature cotrol method (explaied below). Eter the reactio volume ad press «Proceed». The cursor moves over to the «Ru» optio. However, before you ru the protocol, you may select to VIEW the protocol steps, by selectig «View» ad pressig «Proceed». View: Lid:100 C iprf1kb Vol: 20µl 1= 95.0, 2:00 2= 92.0, 0:01 3= 70.0, 0:10 The steps are listed alog with the default heated lid temperature ad selected reactio volume. I this case, you eed to use the «Select» keys to scroll dowward to view all protocol steps. By selectig «Proceed» agai, you are brought back to the previous scree, from which you ca highlight ad select «Ru» to commece the cyclig program. Tip: The MJ Mii thermal cycler comes pre-loaded with a series of template protocols for a variety of commo applicatios (See Appedix A for a full list of protocols). You may chose to ru these programs as they are, or to adjust certai parameters i order to optimize your reactios. Selectig Calculated Temperature Cotrol Mode vs. Block Cotrol Mode With the MJ Mii thermal cycler, the default settig for ruig a protocol is calculated temperature cotrol method. This temperature cotrol method compesates for the fact that the sample s temperature lags behid the block temperature. Whe creatig a ew protocol, you will be asked to eter a sample volume. The cycler will automatically take this volume ito accout whe ruig the protocol ad by employig small temperature overshoots, the cycler rapidly brigs the sample to its target temperature. Every time you select a protocol to ru, you will be asked to eter a sample volume. By eterig zero i the Sample Vol field, the cycler will eter Block Cotrol Mode, this mode does ot employ the small temperature overshoots. As a result, it will take slightly loger for the sample to reach its target temperature ad the protocol will geerally take a little loger to ru. I the U.S., call toll free at BIORAD ( ) Visit o the Web at 27

29 Readig the Rutime Scree Whe a protocol is ruig, a rutime scree will be displayed: Ruig: iprf1kb Step 1: :00 Step 2: :01 Step 3: :10 The scree lists: the protocol ame (iprf1kb i the example above), protocol step that is ruig (Step 1), ad either the block temperature for block-cotrol protocols or the calculated sample temperature for calculated-cotrol protocols. Whe the step s target temperature is reached, a timer starts i the Temp lie (mi:sec). By pressig the Left arrow butto, you ca view the lid temperature. This scree shows oly as log as the left «Select» key is pressed. The rutime scree returs whe you stop pressig the key. By pressig the Right arrow butto, you ca view the total time that the protocol has bee ruig, the total time remaiig i the protocol, ad the cycler umber that the protocol is curretly ruig. This scree is displayed oly as log as the key is pressed. The rutime scree returs whe you stop pressig the key. Readig the Temperature Display Scree By pressig the «Scree» butto, you ca toggle betwee differet screes durig a protocol. Pressig the butto oce will brig you to the temperature display scree: : GOTO x This scree lists a shorthad versio of the protocol. O the left, the list of the temperature steps appears. I this case, the deaturatio temperature (90.0) is followed by the aealig temperature (64.0) as well as a extesio time of This temperature patter is repeated 29 times (as idicated by the GOTO step i the protocol, as well as the 29x show at the bottom of the scree). This cyclig is followed by a fial hold step of O the right, a diagrammatic represetatio of the protocol is preseted. I the example above, the cycler is curretly holdig the samples at 64.0 C. O the left, this step is highlighted, ad o the diagrammatic represetatio this step is blikig. A couter o the top lie of this scree marks the amout of time that has passed o this step (mi:sec). 28 Visit o the Web at I the U.S., call toll free at BIORAD ( )

30 Ruig Protocols Time Remaiig Scree Press the «Scree» butto oce more, to see the time remaiig i the protocol: 00:25:55 Time Remaiig The time is oted i hours:mi:sec. I this case, there are 25 miutes ad 55 secods remaiig util the program is complete. This scree is particularly useful i lab settigs where multiple cyclers are beig ru. From the Time Remaiig Scree, pressig the «Scree» butto returs the user to the Mai Meu. This is the same scree that we have ecoutered previously, except that the bottom lie idicates that the thermal cycler is egaged i ruig a protocol. RUN VIEW NEW FILES EDIT TOOLS Ruig: iprf1kb Readig the Protocol Completio Scree Whe the protocol fiishes, a log beep souds ad the Mai Meu appears. From this Mai Meu, you may select the «Scree» butto to review the parameters of the protocol that just fiished ruig. LAST RUN: iprf1kb HOTLID: 99,30 VOLUME: 20 ELAPSED: 8:47 ERRORS: Noe SOFTWARE: v.1.1a Certai error messages may also be displayed i this scree (see chapter 10). Press «Proceed» to retur to the Mai Meu. I the U.S., call toll free at BIORAD ( ) Visit o the Web at 29

31 Maually Steppig Through a Protocol Durig a protocol ru, pressig «Proceed» allows you to advace the protocol to the ext programmed step, eve if the machie is curretly rampig the block s temperature (see chapter 6 for iformatio o rampig). A cofirmatio scree will be displayed: Ruig: iprf1kb Step 1: :25 Temp: :10 Cycle 2 of 39 Goto ext step? Yes Select Yes, the press «Proceed». The protocol will advace to its ext step. Pausig a Ruig Protocol Press «Pause» to temporarily stop a ruig program. The clock will be replaced with the word Pause o the rutime protocol scree: Ruig: iprf1kb Step 1: :25 Temp: 90.0 PAUSE Cycle 2 of 39 The samples are held at the displayed temperature util either the «Pause» or the «Proceed» key is pressed, which causes the protocol ru to resume. A protocol caot be paused before the target temperature for a give step has bee reached. If «Pause» is pressed before this poit, the block cotiues heatig or coolig util the target is reached, ad the the protocol is paused. 30 Visit o the Web at I the U.S., call toll free at BIORAD ( )

32 Ruig Protocols Stoppig a Ruig Protocol Press «Cacel» to stop a ruig protocol. A cacellatio cofirmatio scree will be displayed: Ruig: iprf1kb Step 1: :25 Temp: :10 Cycle 2 of 39 Cacel iprf1kb Yes Select Yes, the press «Proceed» to cacel the protocol. The total ru time for the protocol will be displayed: PROGRAM CANCELLED Total Time: 3:21 Press «Proceed» to retur to the Mai Meu. Note: If the cycler is curretly rampig to a temperature, the thermal cycler will cotiue rampig util it reaches its target temperature at which poit, you will be able to cacel the program. Note: Turig off the machie does ot stop a ruig protocol. The MJ Mii cycler will assume the protocol was stopped by a power outage ad will resume ruig the protocol whe the machie is tured back o (see below). I the U.S., call toll free at BIORAD ( ) Visit o the Web at 31

33 Resumig a Protocol After a Power Outage If a power failure occurs whe a protocol is ruig, the MJ Mii cycler will hold the protocol i memory for a miimum of 24 hours to a maximum of 7 days, depedig o evirometal coditios. Whe power is restored, the protocol will begi ruig from the poit at which it was iterrupted, ad a otice about the power iterruptio will be displayed. The otice will idetify the step ad the cycle at which the power failure occurred, as well as the block s temperature at the time power was restored: Ruig: A/C POWER FAILED CYCLE 1 STEP 1 RECOVERED AT 46.8 PRESS PROCEED TO CONTINUE Press «Proceed» to remove this scree. The protocol s diagrammatic scree will immediately be displayed. Usig the Istat Icubatio Feature The MJ Mii cycler may be coverted to a costat-temperature icubator by pressig «Istat». A scree allowig use of the heated lid will be displayed: INCUBATE: Use heated lid? Yes Use the «Select» keys to eable or disable the heated lid, the press «Proceed». A scree allowig etry of the icubatio temperature will be displayed. Icubate to Use the keypad to eter ay icubatio temperature from 0 C to 99.9 C, the press «Proceed». The thermal cycler will icubate the sample at the specified temperature util «Cacel» is pressed. 32 Visit o the Web at I the U.S., call toll free at BIORAD ( )

34 Ruig Protocols Icubatig to: 75.0 C STEP 1:75.0 FOREVER TEMP: 75.0 Preheatig Lid Whe the sample block reaches the icubatio temperature (ad whe the heated lid achieves the set temperature), a timer begis ruig o the scree. To stop ad start the timer, press «Pause». Tip: The Pause feature is useful if you eed to temporarily remove samples that must be icubated for a precise period of time. Pausig the timer while samples are ot i the block allows you to track the exact duratio of their icubatio. I the U.S., call toll free at BIORAD ( ) Visit o the Web at 33

35 34 Visit o the Web at I the U.S., call toll free at BIORAD ( )

36 6 Creatig Programs The Elemets of a Program Desigig a New Program Eterig a New Program Modifyig a Program Step with the Optios Revisig Durig Programmig Deletig a Program Keepig a Permaet Record of Programs Selectig a Heated Lid Optio

37 The Elemets of a Program MJ Mii cycler programs cosist of a series of steps ecodig a protocol. These steps are ru usig oe of two possible temperature cotrol methods: calculated cotrol or block cotrol. Programs may cotai four types of steps. Two of the steps are madatory, ad two are optioal: 1. Temperature step (madatory): Sets a temperature for the sample block as well as the legth of time it is held at that temperature. The MJ Mii cycler brigs the block to this temperature at its maximum rate of heatig or coolig, uless modifyig istructios are added to the program. With Calculated Temperature Cotrol Mode, the cycler will icorporate small temperature overshoots to rapidly brig the sample to its target temperature these overshoots, however, do ot alter the target temperature or the icubatio time for a sample. With Block Cotrol, the heat pump brigs the sample holder rapidly to the target temperature (without ay temperature overshoots); samples, therefore, take a little loger to reach their target temperature ad icubatio steps are geerally legtheed to accommodate this. 2. Gradiet step (optioal): Allows you to program a temperature gradiet from frot to back alog the sample block. The rage of ay sigle gradiet ca be as great as 16 C. The maximum programmable temperature is 99 C; the miimum programmable temperature is 35 C. 3. GoTo step (optioal): Causes the program to cycle back to a earlier step for a specified umber of times (up to 9,999 times). 4. Ed step (madatory): Istructs the MJ Mii cycler to shut dow its heat pump because the program is complete. Additioal istructios, termed optios, ca be added to certai program steps to modify their effects: 1. Icremet: Modifies a temperature step to allow a progressive icrease or decrease of temperature ( C per cycle) each time the step is executed i a cycle. This is useful i touchdow programs, whe the aealig temperature of a oligoucleotide is ot kow. 2. Exted: Modifies a temperature step to allow progressive legtheig or shorteig of a temperature step hold (by 1 60 sec/cycle) each time a step is executed i a cycle. This is useful for accommodatig a ezyme with dimiishig activity. 3. Beep: Modifies a temperature step to make the machie beep whe the target temperature is reached. Desigig a New Program Traslatig a Protocol ito a Program Util you are completely familiar with programmig the MJ Mii cycler, you may fid it helpful to first traslate the protocol ito program steps ad optios o paper. Write dow the protocol to be programmed, oe step per lie. The write the type of program step that goes with the protocol steps, at the ed of each lie. If a protocol step ivolves a optio as well as a program step, write both ames dow o the same lie. Fially, write the Ed step at the bottom of the list; programs will ot ru without this step. Number the lies 1 through N, where N is the fial, Ed lie. 36 Visit o the Web at I the U.S., call toll free at BIORAD ( )

38 Creatig Programs Usig the GoTo Step to Write Short Programs The GoTo step allows programs of may repetitious steps to be shorteed to just a few lies. Whe the program ecouters a GoTo step, it returs to a specified step, repeats that step, ad repeats all steps that follow, back to the GoTo step. Whe the program has retured, or cycled, back to the step a specified umber of times, the program moves o to the step that follows the GoTo step. For example, cosider a basic cycle sequecig protocol cosistig of 30 repeats of a deaturatio, ad a aealig/extesio step. Rather tha listig all 60 steps, use a GoTo step to desig a short, easy-to-eter program: Raw program: for 30 sec for 3 mi for 30 sec for 3 mi for 30 sec for 3 mi for 30 sec [cotiues for total of 60 lies] Thermal cycler program: for 30 sec for 3 mi 3. GoTo step 1, 29 times (i.e., cycle back to step 1 ad repeat steps 1 ad 2, 29 times) 4. Ed Choosig a Temperature Cotrol Method The MJ Mii cycler ca cotrol block temperature i two possible ways, ad both have differet implicatios for the speed ad accuracy of sample heatig: Calculated cotrol: The thermal cycler adjusts the block s temperature to maitai samples of a specific volume i a specific vessel type at programmed temperatures. This icludes optimized overshoots of the block by a few degrees for a few secods, which brig the samples to the programmed temperatures. Block cotrol: The thermal cycler adjusts the block s temperature to maitai the block at programmed temperatures, idepedet of sample temperature. Calculated Cotrol Calculated Cotrol is the method of choice for most types of programs, yieldig the most cosistet, most reliable, ad fastest programs. Whe usig calculated cotrol, the thermal cycler maitais a ruig estimate of sample temperatures based o the block s thermal profile, the rate of heat trasfer through the sample tube or slide, ad the sample volume or mass. Sice this estimate is based o kow quatities ad the laws of thermodyamics, sample temperatures are cotrolled much more accurately tha with Block Cotrol. Hold times ca be shorteed sigificatly whe protocols are ru uder Calculated Cotrol. I additio to the simple coveiece of spedig less time ruig reactios, shorter protocols also help preserve ezyme activity ad miimize false primig. Cyclig deaturatios ru uder Calculated Cotrol are usually optimal at 5 secods. Aealig/extesio steps ca also be shorteed, but the periods for these will be reactio specific. I the U.S., call toll free at BIORAD ( ) Visit o the Web at 37