GeneQuant User Manual
Contents
1. Fnsureall parameters in set uo are appropriate for your sarple todisplay absorbance reading Press select tocyclethrough the 4wavelengthvalues 230nm 260nm 280nm and 320nm indicates that acell pathlength of 10mmhas not been selected To convert to corresponding OD values fora 10mmcell multiply by 2 100r20 for5 1and0 5mpathlengthcells respectively todisplayA A absorbance ratio with or without background correctionat 320m See setup to display concentrations of RNA dsDNA or ssDNA Press select tocyclethroughchoiceofunits Conc1 ug ml range 1 4000ug ml Conc2 pug ul rang 0 001 0 24g u1l Conc3 pmol ul range0 001 200pmol u1l Note ensurethe oligonucleotide lengthandfactor valuearecorrect See set uo Conc 4 Phosphate concentrationpmol range 0 001 0 200emoL todisplay protein concentration inmg ml range 0 600mg ml Note ensurethecoefficientsarecorrect See setupo todisplaymolecules ml Note ensurethecorrectmolecularweight isentered See setup todisplayperoentage purity by comparingactual ratiotoexpected ratio Note ensuretheexpectedratioisentered See setup tocalculate percentage recovery by comparing actual toexpected conesntration arol 11 Note ensuretheexpectedconcentrationandthe oligonuclectide lengthareentered See setup does not apply toprinter output version Issue 03 01 2000 English 3 m 3 tocalc2. of Abs reading or mA which ever is the greater 0 to 3 000 Abs 1 of reading or 0 005Ato 3A whichever isthegreater 0 5mmpathlength capillary cell 1mm 5mmand 10 mm pathlength standard ll 270 w x320 d x130 h 3 5kg Indoor use only away from inflammable materials Temperature 5 C to 40 C Maximum relative humidity 80 up to 31 C decreasing linearly to 50 at 40 C InstallationCategory II IEC 1010 CISPR 22 Designed andmanufactured in accordance with an ISO 9001 approved quality system Centronicsparallel output erinterversicnonly 100 120V 1 25AT 200 240V 630 mA T F101 3 15AT F102 2AT 100 VA 100 120 V 200 240 V 50 60 Hz Specifications aremeasuredafter the instrument has wamedup at constant ambient temperatureandaretypical of aproductionunit Aspart of ourpolicy of continuous develoament we reserve the right toalter speci ficationswithoutnotice 20 English Issue 03 01 2000
3. prol phosphate Nucleotide concentration g ml B15 Molecules ml concentration ug ml x 6 023 x 10 MW x 10 Protein mg ml 1 55x A A 0 76x A A Coefficients 1and2 defaults 1 55and0 76 respectively canbe changed for differentproteins Tm1 for short oligonucleotides 2 nA nT 4 nG nC nrefersto nunber of individual base units equation is valid linearly from10tol18mer but may be used as a guideline for values above 18 mer Tmprimers 81 5 16 6 logmolarity 0 41 Sguanosine cytosine 500 primer length Equation is valid from14 to 60mer applies toprinter output versiononly Issue 03 01 2000 English 17 MAINTENANCE Observe all necessary precautions if dealingwithhazardous samples or i ae lets Usermaintenance is restrictedtochangingthe instrument larp changingexternal fuses andinstrument cleaning For any othermaintenance operation contact your localsupgelier Cleaning and General Care External Cleaning Switch off the instrument anddisconnect the power supply cord Useasoft dampclothtocleanall external surfaces Amildliquiddetergent e g Decon maybe used to remove stubbornmarks Sample Compartment Spillages Switch off the instrument anddisconnect the power supply cord The sample compartment is coatedinachemical resistant finish However strongcon
4. oracalculation re 4080 to switch facilityon The instrument will thendisplay Locked key in 4080 againtounlock The instrument remains locked lt isdisplayed keyin Instrument function after a sarp throughpower of f on fli 1 l ly Calculationkeys except Tm will only f Dog measurement has been taken Le Issue 03 01 2000 Instrument Set Up If youkey inanunber incorrectly press C andstart again tosteothrough set up options andenter theparameters which relatetoyour sample Toexit set up press any calculationkey Toreturntothebegimniing press setuo set up enter The default parameters used inthe instrument maybe alteredas follows Cell pathlength mm Press select tochoose from 0 515 10 Printer Press select tochoose franON OFF Press enter Samplenunber Key inthe requirednumber Press enter The sample number increments automatically each time the sample is measured Date Key inthedate Press enter Adjust thisdaily Month Select agorooriatemonth Press enter Year Key intheyear Press enter Background compensation 320mm Press select tochoose from YES NO ADilute Key inthedi lution factor for concentration calculation Range 1 00 99999 9 Factor Press select to choose from RNA dsDNA ssDNA For synthetic oligonuclectides use ssDNAandkey innew factors if defaults arenct suitable Press enter B
5. 4 459395 Exp 15 MELTING TEMP 40 C MELTING TEMP PRI 48 5 C Routineanalysis ofproteins Operator Date 23 Feb 1993 Sample No 8273 PROTEIN CONC 0 7 mg ml Operator Date 23 Feb 1993 Sample No 8274 PROTEIN CONC 0 4 mg ml Operator Date 23 Feb 1993 Sample No 8275 PROTEIN CONC 1 5 mg ml Operator Date 23 Feb 1993 Sample No 8276 PROTEIN CONC 3 8 mg ml Operator Date 23 Feb 1993 Sample No 8277 PROTEIN CONC 32 2 mg ml Operator Date 23 Feb 1993 Sample No 8278 PROTEIN CONC 0 2 mg ml Issue 03 01 2000 13 Parallel Printer Interface Specifications Datatransmission 8bitparallel Synchronisation Timing for attachedprinter isprovidedby external strobe signals Handshakeprotocol By BUSY and STROBE signals Signal levels The levels of output data and interface control signals areall TILcompatible Interface Connector Theprinter interface isastandard25 pinD shell femaleconnector Thedata Lines DO D7 onthe connector are driven by drivers capable of sourcing 15mA and sinking 24nA ga 5 5 S Da Da Da Da Da Da Da Da O 0 au a OT e a o H HA O OOQ O OOOO m Y ha zZ D Signal Name STROBE taBit 0 taBit 1 taBit 2 taBit 3 taBit 4 taBit 5 taBit 6 taBit 7 ACK BUSY Unconnected 18 25 N A Ground 14 English Issue 03 01 2000 ACCESSORIES AND CONSUMABLES Selecting th
6. CONTENTS Page 2 3 Section INTRODUCTION SYMBOLS REAR PANEL BEFORE INSTALLATION INSTALLATION CALCULATION KEYS Other Keys OPERATION Instrument Set Up Reference Measurement Sample Measurement Printer Output Version ACCESSORIES AND CONSUMABLES Selecting the Appropriate Cell UV Visible Cell Order Information Using Cells Other FACTORS AND FORMULAE MAINTENANCE Cleaning and General Care Fuse Replacement Deuterium Lamp Replacement SPECIFICATIONS Issue 03 01 2000 English INTRODUCTION GeneQuant the RNA DNA calculator is aspectrophotometer designed specifically formoleailarbiologists The instrument measures RNA and DNA samples in UV cells at 230 nm 260 nm 280 nmand 320 nmsimultaneously The wavelengths 260 nmand 280 nmare used for quantification andpurity checking calculations whereas 320 nmcanbe used for background compensation 230 nmcanbe usedas a guide for protein determination using the peptide bondabsorbance After each sample reading the infomation is storedandusedincalculations until thenext reading is taken GeneQuant canbe left on continuously without affectingthe lifetime of the light source andthis is recommended for the convenience of the user to obtain rapid measurements The nomal status of the lamp is standoy mode and it is only powered up during actual reference and samplemeasurement using a unique demand switchingtechnique Patent agpliedfor You ca
7. ally displays the result after samplemeasurement displayedonprinter output versiononly 10 English Issue 03 01 2000 Printer Output Version This section shouldbe readinadditiontothe details given intheprevious sections Installation Connect theprintervia its interface cabletothe socket onthe rearpanel of the instrurent Switchontheprinter andensure it ison Line Operation The sample nunber anddate are storedpenmanent ly until altered Sample nunber is incrementedautomaticallyeachtimea sample is measured The date does not increment automatically Tf youdonct wishtoalter any further parameters in set up take reference readings and samplemeasurements as describedpreviously Thedisplays and printouts appear as shownbelow whentheprinter option in setup issetto on printouts of thedisplayococurautaratically After youhave removedthe reference ABSORBANCE Absorbance 260 nm 0 000 AU 260 nm 0 000 AU select ABSORBANCE Absorbance 280 nm 0 000 AU 280 nm 0 000 AU The remaining absorbance values canbe printedout inthe same way Issue 03 01 2000 English 11 When a sample is measured sample Operator Soe ied Date 23 Aug 1993 Sample No 9164 Insert Sample Remove Sample ABSORBANCE Absorbance 260 nm 0 957 AU 260 nm 0 957 AU RNA DNA dsDNA CONC 1 dsDNA CONC 1 47 9
8. ases Key inthe number of AC GT U to calculate molecular weight frombase composition of nucleicacids andto showresultsasmolecules ml Press enter tocyclethroughthebases Range 0 1000 Oligolength Key inthe oligonucleotide length of the sample inbase units toshowresultsaspmol 1 Press enter Range1 9999 Molecularweight MN Press select to choosebetween the calculated value fromA C G T Unumbers or user enteredvalue formolecular weight ifknown Press enter Ratio Key intheA A absorbance ratio expected for your sample if known Press enter Concentration Key inthe concentration expected inpwles ul for your sample ifknown Press enter Protein Key incoefficients forequationif theyaredifferent todefaults See Factors andFommilae Molarity Key inmolarityof salts inhybridisationsolution apoliestoprinter output versioncnly does not apply toprinter output version 8 English Issue 03 01 2000 Reference Measurement Take a reference reading This reading is storedandusedas the base reading for all samplemeasurements until anew reference readingis taken If youdonct insert and remove the sample cell intir thedisplaywill Remove Cell Press Key Again show set Please ref Wait 2 Wait fort
9. centrations of samplemay affect the surface andspillages shouldbe cealt with immediately Asmall drainhole inthe sample campartment allows excess liquidtodrain away onto thebench or table fromunder the instrument Use a soft dry cloth tomop out the sample compartment Reconnect the power supply cordand switch onthe instrument Fuse Replacement Select the appropriate fuses for your local supply Two identical fuses needtobe loaded For LO 100 120V operation use 2 x 1 25AT fuses and for HI 200 240V operation use 2 x 630mAT fuses Switch off the instrument anddisconnect the power supply cord The fuse holder can only be opened if the power supply plug has been removed The fuse holder is locatedbetween the power input socket andthe on off switchontheback panel of the instrument Slide openthe fuseholderbypullingat thenotch Place fuses intothe fuseholderandslideback intoposition Reconnect the power supply cordand switch onthe instrument 18 English Issue 03 01 2000 Deuterium Lamp Replacement Replacement lamps are available franyour local supplier Th The deuterium lampbecames very hot inuse soallowat least 10 i ff minutes before changing Careshouldbetakennot totouch theq tical ra surface of thenew Lamowith your fingers if touched thearea shouldbe cleanedwithmethanol Switchoff the instrument anddis
10. connect the power supply cord Release the instrument coverby unscrewing the seven screws inthebase Carefully lift toocoverupwards tilt andplace onthe right sideof the instrument taking care not todamage the ribbon cables Depress catch A and lift connector away fromcircuit board Remove two screws B Lift deuterium lamp andbracket assembly away frommountingplate Place newdeuteriumlampandassembly intoposition locatingpins C into holes andslot inmountingplate Refit two screws B andtighten Refit connector A pushing downwards until the catch snaps shut Refit the instrument topcover takingcarenot totrapthe rilboncable Refit the seven screws inthebase Reconnect the power supply cordand switch onthe instrument Issue 03 01 2000 English 19 SPECIFICATIONS Light source Wavelength range Wavelength calibration Wavelength accuracy Wavelength reoroducibility Bandwidth Straylight Photaretricreoroducibi lity Photometric range Photometric linearity Cell size accommodated Dimensions Weight Environmental conditions Safety standard EMC standard Quality system Digital Output El ical Ficti Extemal fuseratings Interal fuseratings VArating Voltage Frequency Deuteriumarc lamp Fixedat 230 260 280 and 320 nm Factory set 2 nm Better than 0 1nm 5 nm Less than 0 1 T at 320 nmwith NaNO 0 5
11. e it can beenptiedusinga Pasteurpipettebulb attachedtonarrowbore silicone tubing The quartz capillary cell canbe dismantled for cleaning and removing a broken capillary by unscrewing the two screws oneach side usingthe tool provided Instructions areprovidedwith the 0 5amquartz capillary and Smultra microvolumecells Other GeneQuant GeneQuant printer version GeneQuant II Deuterium lamp GeneQuant User Manual GeneQuant II User Manual Dust Cover Parallel InterfaceCable Centronics Introduction toBasicUV Visible Spectrophotamretry 80 2103 98 80 2104 98 80 2105 98 80 2104 56 80 2105 20 80 2105 58 80 2105 18 80 2071 87 80 2005 60 16 English Issue 03 01 2000 FACTORS AND FORMULAE Grams g are convertedtomoles using 309 the averagemolecular weight of the ATCG bases Factors formolecularweights MW of bases A 312 2 C 288 2 G 328 2 T 303 2 U 289 2 For dephosphorylatedoligonucleotides subtract 61 fromthe calculated MW Forphosphorylatedoligonuclectides add17 tothe calculated MW B X factor g ml concentration Default factors are 40 for RNA 37 for ssDNA 50 for dsDNA For synthetic oligonucleotides use ssDNAmode and change factor A o a ratios are 1 8 and2 0 for pure DNA and RNA preparations respectively pmol ul ug ml x 1000 pmol W1 Ug ml x 1000 309 xoLigo length MW
12. e Appropriate Cell GeneQuant has a suitable measuring range between 0 1to2 50D fora 10mm pathlengthoell Choosea suitable cell dependingon sample concentration range dilution factor andavailable samplevolure Pathlength mm 10 5 1 0 5 Suitable Sample Concentration range lg ml giventhat OD2 9 range 1 0 OD260 50ug ml for dsDNA 2 5 0 1 125 5 5 0 2 250 10 25 1 0 1 250 50 50 2 0 2 500 100 UV Visible Cell Order Information Pathlength andDescription Minimum Sample Volume Part Number 10m standardcell with lid 2 00QU1 80 2002 58 10mm semi microcell with lid 50Qu1 80 2002 77 10m microcellwithlid 25041 80 2002 95 10m ultramicrovolume cel TUL 80 2103 69 5m standardcellwithlid 1 00QUL 80 2002 57 5m ultramicrovolume cel Gul 80 2103 68 Im standardcellwithlid 200QUL 80 2002 54 0 53m quartz capillary l ul 80 2104 66 incluces 100 capi aries Sparequartz capillaries 100 pL 80 2104 67 Issue 03 01 2000 English 1 5 Using Cells Ensure that the cell faces are cleanbeforemeasurement Afteruse cells shouldbe cleanedwithadilutealkali e g 0 1MNa0H andadiluteacid e g 0 1MHC1 wash followedby rinsing several times withdistilledwater More rigorous cleaningafter difficult samples shouldbeperformedwitha suitable liquid detergent followingthemanufacturer s instruction The 0 Smmquartz capillary is filledby dipping intothe sample Afterus
13. fanoutlet isnot dostructed INSTALLATION at li This equipment must be connected to the power supply with the power la supply cord provided and MUST BE EARTHED fos k Ifthisequipent is used inamannernot specifiedor inenvironmental conditions not agerooriate for safeqoeratiion theprotecticonprovidedby the equipment may be impaired and instrument warranty withdrawn Select the correct voltage for your local supply usingthevoltage selectoronthe rearpanel Select the agoropriate fuses for your local supply Two identical fuses needtobe loaded For LO 100 120V operation use 2x1 25AT fuses and for HI 200 240V operation use 2 x 630mA T fuses SeeMaintenance for fitting fuses Connect the power supply cordtothe input socket onthe rear panel andto the power supply Switchonthe instrument Forootinumlocationof thecell repositionthe springclip located inthe sample compartment by pulling firmly upwards and relocating inapgpropriatecentral slots andif youarenot using 10mmcells reset thepathlengthin setuo Tf youareusingaprinter check that it isaparallel versicnandis switched on Line ifnecessary Ensurethat theprinterogotions are selectedin setap applies toprinter output versiononly 4 English Issue 03 01 2000 CALCULATION KEYS abs ratio RNA DNA protein Ix purity recovery
14. hetoneandinsert Insert the referencece11 intothe Reference sample compartment 3 Wait forthe tone and remove Remove thereference Reference 4 After youhave removed the ABSORBANCE referencethedisplaywill show 260 nm 0 000 AU Whenusingthe capillary cell andholder placethe holder inthe sampl compartment before takingmeasurements Then insert and remove the capillaryasabove Issue 03 01 2000 English 9 Sample Measurement You can nowmeasure a sample A similar procedure is followed to the one used formeasuringa reference Usethe sample key insteadof set ref 1 sample Wait forthe tone and insert the samplecell into the sample compartment Wait forthe tone and remove the sample cell Afterthe first sample the displaywill show absorbance at 260nm Ensure correct cell pathlength andsample type have been selectedin set up Please Wait a Date 23 Aug 1993 Sample No 9164 Insert Sample Remove Sample ABSORBANCE 260 nm 1 000 AU Absorbance indicates that a 10mm pathlengthcelLhas not been selected See setuo Stored readings can nowbemanipulatedusing the calculation keys For subsequent samples the display defaults totheprevious function used This facilityprovides useful single key operation if you require the same type of measurement on successive samples Operationof the sample key autamat ic
15. n use GeneQuant to calculate RNA ssDNA anddsDNA concentrations inunits of weight molar fraction moles of phosphate andtotal molecules Bo B ao ratio Total proteinconc entration Recoveryofoligonucleotides Purityofnucleicacids Meltingtemperature Molecularweight of oligonucleotides 2 English Issue 03 01 2000 SYMBOLS Symbols found in the manual Instrument l PleaseNote Ready Display Panel fy fi sampl 1 ft ple l ee Warning Key Press 1 2 toad Symbols foundon the instrument Background yellow symbol black Caution refer to accompanying documents o REAR PANEL Mains Power Switch p ON O OFF Voltage Selector Cooling Fan Outlet LO 100 120V ATAN HI 200 240 a ae Ae Co Ga O a Ge SS a oe Fuse Holder PRINTER o o00c0 000000 9000000600000 a ZD Co co ces co cer Co CO 630mA T HI Power Input Socket Printer Socket Printer Version Only Issue 03 01 2000 English 3 BEFORE INSTALLATION Inspect the instrument for any signs of damage causedintransit If any damage is apparent then informyour supplier immediately anddo not proceedwith the Check that the installation site conforms totheenvironmental conditions for safe qperation seeSpeci fications thecooling
16. ug ml 47 9 ug ml If requiredpress select tocyclethroughandprint out the other choicesofunits Other calculations areprintedout autamaticallywhenthe appropriate key is pressed The instrument facilitates routineanalysisasthedisplay andprint out always showthe selectedparameters autamaticallyaftera measurement as shown inthe examples on the followingpage Iftheprinter is switchedoff or off lineduringusewhile selected on in set up andakey ispressed thedisplay shows Printer off line Press any key Nommal functioning resumes after any key ispressed Iftheprinter is switchedon again nonrmalprintingresumes Iftheprinter remainsoff theprintergotion in set up isautaraticallyset tooff 12 English Issue 03 01 2000 setuo tions GeneQuant SETUP Path Length 10 Printer ON Sample No 507 Date 18 Month Jan Year 1993 Use 320 nm NO Dilute 1 00 FACTOR ssDNA 37 0 BASES Number A 5 Number C 5 Number G 2 Number T at Number U a OLIGO Length T3 MOLECULAR WEIGHT CALC 3961 6 RATIO Expected 1 800 CONCENTRATION Expected 2 000 PROTEIN Coeff 1 1 550 Coeff 2 0 760 Molarity 0 100 Canplete list of calculations fora nucleotide Operator Date 18 Jan 1993 Sample No 0505 ssDNA CONC 1 29 3 ug ml ssDNA CONC 2 0 029 ug ul ssDNA CONC 3 0 197 pmol ul Phosphate CONC 0 093 pmol RATIO 1 997 PURITY 99 RECOVERY 98 MOLECULES ML
17. ulatemeltingtemperature Press Select tocyclebetween shortoligonucleotideandprimercalculation Note ensurethat thenumber of bases and molarity are correct Se setup indicatesthat theequationisnot strictlyagolicable forthehase nunbersused SeeFactors andFomnlae O my 49 I A lt Wn factor enter select set ref sample n N to change factor values for RNA ssDNA and dsDNA and select the current moce for calculations toenteravalueor option tochoosea value or optionwithina function tomeasure reference tomeasure sample toprint out the List of set uo tions undefined available for futuredevelomment applies toprinter output versioncnly Oy English Issue 03 01 2000 OPERATION Toget started Instrument Initialising Instrument Ready set reference measure your sample switchonthe instrument at the rearpanel Note Insi trument initialises fora fewseconds select set uo values This steocanbe anitted for quick absorbance readings insert thecel sothat the Lightpathdirectionis inthe front to rearaxisofthe instrument as indicatedby the arrow The instrument canbe useddirectly as acalculator for Tmand molecular weights without measurement of a sample Asecurity facilitywhich locks the instrument keypadis available When Instrument Ready
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