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INFORMATION FOR

Mapping

Enzyme Selection and Primer Design for Gene Mapping Protocol

Protocol in Word Format

1.Use Wat-Cut to identify restriction sites in sequences containing SNPs

http://watcut.uwaterloo.ca/watcut/watcut/template.php

2.Under “SNP-RFLP analysis” tab, paste in your sequence and Submit.

You will get a list of possible restriction sites.

  • Choose your enzymes by:
    • Price: some enzymes are much more expensive. See table this page.
    • Availability: nice to use commonly available enzymes
    • Enzymes not requiring any base changes in the primer sequence to work

Watcut allows you to save your preferences, saving time.

Enzyme

Price/Unit

BamHI

$0.005

HindIII

$0.005

ecoRI

$0.005

pstI

$0.006

pvuII

$0.011

apaI

$0.013

XhoI

$0.013

EcoRV

$0.013

kpnI

$0.015

NdeI

$0.015

styI

$0.021

XbaI

$0.021

bglII

$0.027

avaI

$0.028

avaII

$0.028

sacII

$0.028

draI

$0.029

ncoI

$0.056

xmnI

$0.056

stuI

$0.056

DraIII

$0.061

accI

$0.066

AatII

$0.106

NarI

$0.116

speI

$0.122

sphI

$0.122

pvuI

$0.126

AgeI

$0.244

nspI

$0.244

psiI

$0.525

3.Use Primer 3 to design your Primers

  • http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi
  • Primer3 Plus Settings:

    General Settings:

    Product size range: 100-250

    Min size: 18

    Opt size: 20

    Max size: 22

    Min Tm: 55

    Max Tm: 65

    Min GC%: 45

    Max GC%: 75

    Advanced Settings:

    Max PolyX: 3

    CG clamp: 2

  • Paste your sequence into the input field
  • Identify your SNP, and select 50 bp flanking either side of SNP. This will ensure the primers will flank your SNP by a sizable piece of DNA, making it easier to resolve the RFLPs on a gel.Use the square brackets to select the area to be amplified, and click “Pick Primers”.

Prepare your DNA if necessary. See DNA Isolation Protocol .

Amplify your sequence for each sample, and check for DNA using 10ul sample on regular 2% agarose gel. Refer to PCR and Restriction digest for SNP Detection protocol for details.

Word

Digest your samples using appropriate enzymes.

  • Digest samples 4 hours (if in PCR machine), or up to overnight(if in incubator)
  • Run out entire digest, along with 10 ul undigested DNA on 4% 50:50 (NuSeive:regular agarose) gel. See Preparing 4% Gel Protocol on our website, or view as Word.