Gradient PCR - wewtfreyghtwyhrtjuhryjryg PDF

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Primer Optimization Using Temperature Gradient Protocol PCRTechnol ogi esPr ot ocol sTabl eofCont ent s

Optimization of qPCR Conditions Opt i mi z at i onofqPCRc ondi t i onsi si mpor t antf ort hedev el opmentofar obustassay .I ndi c at i onsofpooropt i mi z at i onar ea l ackofr epr oduc i bi l i t ybet weenr epl i cat esaswel l asi neffici entandi nsens i t i v eas say s.Thet womai nappr oachesar e opt i mi z at i onofpr i merconcent r at i onand/ oranneal i ngt emper at ur es . Oneappr oac ht oas sayopt i mi z at i oni st odet er mi net heopt i mum anneal i ngt emper at ur e( Ta)oft hepr i mer sbyt es t i ng i dent i calr eac t i onscont ai ni ngafix edpr i merconcent r at i on,acr ossar angeofanneal i ngt emper at ur es.Thi scanbeachi ev ed i faqPCRi nst r umentwi t hat emper at ur egr adi entbl ocki savai l abl e.I nt hef or matpr esent edi nt hi spr ot oc ol ,pr i mer sar e i nc l udedatafi nalconcent r at i onof450nM,andt hegr adi enti sor i ent at edacr os st heXaxi soft hebl ocksucht hatal l col umnsar es ubj ect edt ot hesameTa ( i . e. ,12di ffer entt emper at ur es) .I not heri nst r ument s,t hegr adi enti sdownt hecol umn sucht hatal lr owshavet hesameTa( i . e. ,8di ffer entt emper at ur es) .Thef ol l owi ngpr ot ocolcanbeappl i edt oei t her ,al bei t af t ermi normodi ficat i ons.

Equipment  

Quantitative PCR instrument with integrated gradient block control function Laminar flow hood for PCR set up (optional)

Reagents    

cDNA diluted 1:5-1:10, gDNA 10ng, synthetic oligo (10,000 copies) or other suitable template for optimization. KiCqStart SYBR® Green ReadyMix™ (KCQS00/KCQS01/KCQS02/KCQS03; instrument specific, see Table P1742). PCR grade water: PCR grade water (W1754 or W4502) as 20 mL aliquots; freeze; use a fresh aliquot for each reaction. Forward and reverse primers concentrated stocks (10 μM working stocks: GOI). • Custom oligos can be ordered at sigmaaldrich.com/oligos.

Tabl eP1742.SYBRGr eenPCRMi xSel ect i onGui de.

Hot Start ReadyMixes (Taq, Buffer, dNTPs, Reference Dye, MgCl2)

KiCqStart® SYBR®Green qPCR ReadyMix™, Cat. No. KCQS00

KiCqStart® SYBR® Green qPCR ReadyMix™ Low Rox , Cat. No. KCQS01

KiCqStart® SYBR® Green qPCR ReadyMix™ with ROX, Cat. No. KCQS02

KiCqStart® SYBR® Green qPCR ReadyMix™ for iQ, Cat. No. KCQS03

Compatible Instruments:

Compatible Instruments:

Compatible Instruments:

Compatible Instruments

Bio-Rad CFX384™

Applied Biosystems 7500

Applied Biosystems 5700

Bio-Rad iCycler iQ™

Bio-Rad CFX96™

Applied Biosystems 7500

Applied Biosystems 7000

Bio-Rad iQ™5

Bio-Rad MiniOpticon™

Fast Applied Biosystems ViiA 7

Applied Biosystems 7300

Bio-Rad MyiQ™

Bio-Rad MyiQ™

Stratagene Mx3000P®

Applied Biosystems 7700

Bio-Rad/MJ Chromo4™

Stratagene Mx3005P™

Applied Biosystems 7900

Bio-Rad/MJ Opticon 2

Stratagene Mx4000™

Applied Biosystems 7900 HT Fast

Bio-Rad/MJ Opticon®

Applied Biosystems 7900HT

Cepheid SmartCycler®

Applied Biosystems StepOnePlus™

Eppendorf Mastercycler® ep realplex

Applied Biosystems StepOne™

Eppendorf Mastercycler® ep realplex2 s

Illumina Eco qPCR

Qiagen/Corbett RotorGene® 3000

Qiagen/Corbett RotorGene® 6000

Qiagen/Corbett RotorGene® Q

Roche LightCycler®480

Supplies   

Sterile filter pipette tips Sterile 1.5 mL screw-top microcentrifuge tubes (CLS430909) PCR tubes and plates, select one to match desired format: • Individual thin-walled 200 μL PCR tubes (Z374873 or P3114) • Plates - 96-well plates (Z374903) - 384-well plates (Z374911) • Plate seals - ThermalSeal RTS™ Sealing Films (Z734438) - ThermalSeal RT2RR™ Film (Z722553)

Notes for this Protocol   

cDNA is generated using random priming or oligo-dT priming method and diluted 1:10 for use, but any suitable, alternative template may be used. All reactions are run in duplicate as technical replicates. If using a PCR plate, follow a plate schematic (e.g., shown in Figure P14-19) to ensure that the reaction mix, samples and controls are added to the correct wells.

Method 1. Pr epar eamas t ermi xf or56r eact i onsaccor di ngt oTabl eP1435.Mi xwel l ,av oi di ngbubbl es .

Tabl eP1435.React i onMast erMi xf orTa Opt i mi zat i on.

Reagents

Volume (μL) per Single 20 μL Reaction

Volume (μL) for 56 Reactions

KiCqStart SYBR® Green qPCR ReadyMix 2×

10

560

Forward primer (10 μM)

0.9

50.4

Reverse primer (10 μM)

0.9

50.4

PCR grade water

4.2

235.2

2. Remov e448μLofmast ermi xf r om s t ep1( i . e. ,hal f)i nt oasepar at et ubef orset t i ngupt heNoTempl at eCont r ol( NTC) r eact i ons . 3. Add112μLoft empl at et ot her emai ni ngmast ermi xf r om s t ep2.SetTempl at emast ermi xoni ce. 4. Add112μLofwat ert ot heot herhal foft hemast ermi xf r om st ep2.SetNTCmas t ermi xoni ce. 5. Al i quot20μLTempl at emast ermi xf r om s t ep3i nt ot wor owsoft hePCRpl at el abel edGOI . 6. Al i quot20μLNTCmast ermi xf r om s t ep4i nt ot wor owsoft hePCRpl at el abel edNTC. 7. Cov erpl at esandl abel .( Mak esur et hel abel i ngdoesnotobscur ei nst r umentex ci t at i on/ det ect i onl i ghtpat h. ) 8. Runs ampl esaccor di ngt ot het hr ees t eppr ot ocolbel ow ( Not e:Thesecondi t i onsar especi ficf orFASTc y cl i ngpr ot oc ol s)ens ur i ngt hatt heanneal i ngt emper at ur ehasbeen defi nedonagr adi entbet weent hel owestandhi ghes tt hatwoul dbeappr opr i at ef ort hepr i mer s( ex ampl eshows 54–70° C) .St eps1–3ar er epeat edt hr ough40cy c l es.Ast andar ddi ssoci at i oncur v ei sr unaf t erampl i ficat i on. Tabl eP1436.FASTqPCRCycl i ngCondi t i onsf orTaOpt i mi zat i on.

FAST Cycling Conditions

Temp (°C)

Time (sec)

Initial denaturation/Hot Start

95

30

Step 1

95

5

Step 2

54–70 (gradient)

15

Steps 1–3 are repeated through 40 cycles

Step 3

Not e:Uses t andar ddi ss oc i at i oncur v epr ot ocol( dat acol l ec t i on) .

72

10...