Protocol in Word Format
1.Use Wat-Cut to identify restriction sites in sequences containing SNPs
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.
3.Use Primer 3 to design your Primers
Primer3 Plus 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
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.