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Primer Design and Testing Guide

We are accepting orders from the public. The following are the steps required:

Design Primers for Your Gene

Design primers that amplify 1 kb to 2 kb target TILL fragment and have HIGH (70°C) thermal stability using Primer3. Blast your primers against the genomic DNA to ensure that they target a single sequence. Alternative primer sets are strongly recommended to prevent delays. Fragments with small introns are preferable.

Note: As of December 31, 2013, CODDLE is no longer available. We are working on a command line tool for designing TILLING primers. Please use Primer3 with the conditions listed below in the mean time to design your primers.

Primer3 conditions:

  • Primer Size
    • Minimum: 20 bp
    • Optimal: 27 bp
    • Maximum: 30 bp
  • Primer Temp
    • Minimum: 67°C
    • Optimal: 70°C
    • Maximum: 73°C
  • Product Size
    • Minimum: 1000 bp
    • Optimal: 1500 bp
    • Maximum: 2000 bp

Testing Your Primers

Here are the parameters for our amplification if you would like to test your primer sets in your own lab before sending them to us:

Test your primers on the following species:

  • Arabidopsis thaliana, accession Columbia
  • Solanum lycopersicum, cv. VFNT Cherry (LA1221)
  • Oryza sativa L. japonica, var. Nipponbare

using the the following PCR conditions (Last Updated June 16, 2010):

Reagents Final Concentrations
10X Ex Taq Buffer 1X
MgCl2 2.0 mM (We use Ex Taq buffer that includes Mg+2)
PVP 1.5%
dNTP 0.2 mM
Primers 0.3 µM
Takara Ex Taq Hotstart (#RR006H, 5 units/µl) 0.15 µl/30 µl rxn
Template DNA varies with species (see below)
Reaction Volume 30 µl

Amount of template DNA varies with species as follows:

  • Rice: 1.5 ng
  • Tomato: 2.5 ng


Cycling program

  • 95°C 2 min.
  • Loop 1: 8 cycles
    • 94°C 20 sec.
    • 73°C 30 sec.
    • Increment @ -1°C/cycle (touch down)
    • Ramp to 72°C at 0.5°C/sec.
    • 72° 1 min.
  • Loop 2: 25 cycles
    • 94°C 20 sec.
    • 65°C 30 sec.
    • Ramp to 72°C at 0.5°C/sec.
    • 72°C 1 min.
  • Hold 8°C

We cannot guarantee that primer sets that work in your lab will also work in the TILLING lab. If you do decide to test your primers ahead of time and they work, this is not a guarantee that they will work easily for us. To increase your chances of having primers that will amplify in the TILLING lab, consider creating several alternative primer sets for us to test.

CHANGES from the above protocol

Our high-throughput operation does not allow us to incorporate protocol variations. Due to the fact we are a production facility, please only send us primers that work under TILLING conditions. We do not have the resources to trouble-shoot primers that do not work in our laboratory.

Placing an order

Please refer to our order information page for step by step instructions on how to place orders.

Mutation Detection Approach

We have converted from LiCor-CELI assays for detection of mismatches to next-generation sequencing of pooled genes through the Illumina HiSeq2000 platform.


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