Mapping
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.
WordDigest 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.