LAB 6 PCR - Metodica laboratorio PDF

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ESPERI ENZA 6TDL|PCR Pr ot ocol Equi pmentandMat er i al sRequi r ed           

DNAt empl at e( 1g/l ) Forwar dPr i mer( 5M) Rever sePr i mer( 5M) TaqDNAPol ymer ase( 2U/l ) React i onBuffer5X dNTPMi x( 10mM ofeachdNTP) 5 0 mM MgCl 2 Nucl easeFr eeWat er Ther malcycl er Adj ust abl emi cr opi pet t esandt i ps Mi cr ocent r i f uge

Tabl e1.RecommendedFi nalConcent rat i onsofComponent sf orPCR Component s

Vol ume

Nucl easeFr eeWat er ( t oafinalvol umeof50µl )

Xµl

•React i onBuffer5X

?µl

Fi nalConcent r at i onoramount

1X

5X∙X =1X∙50uL

x=

( 1 x 50 ) ume=10uL =10uL Vol 5

•dNTPmi x( 10mM ofeachdNTP) ?µl

200M

10mM =10. 000uM 200uM ∙50uL=10. 000uM ∙X

X=

200 x 50 =1 uL 10000

Vol ume=1uL •For war dPr i mer( 5M) Rever sePr i mer( 5M)

?µl ?µl

0. 3µM 0. 3µM

10uM ∙X=0, 3uM ∙50uL

X=

0,3 x 50 =1,5uL Vol ume=1, 5uL( perent r ambiipr i mer s) 10

•MgCl 0mM 25 50mM ∙X=1, 5mM ∙50uL

X=

1,5 x 50 =1,5 uL 50

Vol ume=1, 5uL

?µl

1. 5mM

•TaqDNApol ymer ase( 2uni t s/µl ) 1µl 0. 2uni t s I lvol umedi1uLdel l aTaqDNApol i mer asider i vadal l anecessi t adidoverdi l ui r ei l campi oneperpot er l opi pet t ar e( vol umeor i gi nar i o0, 1uL) -f or ni t ogi àdi l ui t o •DNAt empl at e

Yµl

0. 2g

1ug:1mL=0, 2ug:x I lr i sul t at odiquest apr opor zi onesar ebbei mpossi bi l edapi pet t ar e( 0, 2uL)percuiè necessar i odoverdi l ui r ei lcampi onedi10vol t e(1par t ediDNAst ampo, 1uL+9di H2O,9uL) Dalt ot al edi10uLpr el evando2uL,avr emoi0, 2ugnecessar ial l anost r ar eazi one.

Ampl i ficazi one I mpor t ant e:Cambi ar epunt adel l ami cr opi pet t aadogniaggi unt adiuncomponent e del l ami scel af acendoat t enzi oneanoncont ami nar eivar icampi oni . 1.Combi nar eipr i miseicomponent idel l at abel l a1nelt ubodir eazi oneda0. 2mL. Cent r i f ugar ebr evement enel l ami cr ocent r i f uga 2.I ni zi ar el ar eazi oneaggi ungendol ost ampoel aTaqDNApol i mer asi 3.Posi z i onar ei lt ubodir eazi onenelbl occoat emper at ur acont r ol l at aepr oceder econ i lpr ofil odici cl at ur at er mi cascel t aperl ar eazi one.

Thermalcycl i ngprofil e

§

Pr edenat ur at i ont emper at ur e

96° C

5mi n

Denat ur at i ont emper at ur e Annealt emper at ur e Ext ensi ont emper at ur e

94° C 4565° C 72° C

30sec 30sec 60sec

Post ext ensi ont emper at ur e

72° C

7mi n

4° C

hol d

3035cycl es

PCR 1 Not e: For war dpr i mer : 5’ CCGGAATTCCATGCGGGAC– 3’ Rev er sepr i mer : 5’ -GTCCGGCACTCGAGCGCG–3’

Tm= Tm?

PCR A: Not e: For war dpr i mer : 5’ CCGGAATTCCATGCGGGACTACGACGAGGT–3’ Tm=( 18X4) +( 13X2) =102° C Rev er sepr i mer : 5’ CCAGTTGTCCTCAGCCACCAGATTCCACTC3’ Tm=( 17X4) +( 13X2) =94° C

Anal i si : 1.Dopol afinedel l aPCR ( l amaggi orpar t edeipr ogr ammiPCRi mpi eganoci r ca2or e) , anal i z z ar ei lpr odot t odir eazi onet r ami t eel et t r of or esisugeld’ agar osi odi5uLdalt ot al e delpr odot t odir eazi one.I lpr odot t odovr ebbeesser evi si bi l et r ami t et r ansi l l umi nazi one UVsulgeli mmer soi net i di obr omur o 2.Conservar ei lpr odot t odir eazi onea20° C finchénecessar i o.Ipr odot t idir eazi one pot r annoesser eul t er i or ment emodi ficat iusandoal cunepr ocedur ei ncl usal aWi zar d PCR DNAPur i ficat i onSyst em. Comei mpost ar eunTermoci cl at or e Nuovopr ogr amma: ( PROG) ( EDI T) ( NEW) Scegl i er enome,i mpost ar eici cl i : STAGE:1 STEP:1 pr edenat ur azi one STAGE:2

STAGE 3

STEP:1 denat ur azi one STEP:2 Anneal i ng STEP:3 Est ensi one

35ci cl i

STEP:1 POSTEst ensi one

•Mant ener ea4° C quandofini sceperconservar e...