TITLE: Genotyping Arabidopsis T-DNA lines
AUTHORS: Steven J Burgess

[DESCRIPTION]
This protocol is used for genotyping Arabidopsis seedlings to test for the presence of a transfer DNA (T-DNA) insertion. By using two primer sets it is possible to determine whether a seedling is homozygous, heterozygous or azygous for an insertion in the predicted genomic location. 

To identify lines with T-DNA insertions in a gene of interest, you need the Arabidopsis Genome Identifier (AGI) number corresponding to the genomic locus (e.g. RCS1A = AT1G67090), then visit the Salk Institute T-DNA Express site to find all the mapped insertions at your locus of interest. 

Genotyping primers have been pre-designed for each T-DNA line, these can be retrieved from the Salk Institute T-DNA primer site, and ordered at any supplier of DNA oligonucleotides before starting the protocol.

In the US T-DNA lines can be purchased from the Arabidopsis Biological Resource Center (ABRC) and in the UK and EU from the European Arabidopsis Stock Center (NASC).

Recommended reading
http://signal.salk.edu/tdnaprimers.2.html

Setting up the PCR reaction
Genotyping is performed with the Phire Direct PCR Mix, this includes the polymerase, nucleotides and salts necessary for amplification. We use the “dilution protocol” which involves taking a small leaf disk and homogenizing it in dilution buffer using a gel tip (see manufacturer’s instructions for more details.) 

[STEPS]
SECTION: Prepare primer working solution
1. Re-suspend lyophilized primers in dH2O to a stock concentration of 100 mM. Note: primer sequences can be obtained from SALK T-DNA express if you have the T-DNA accession number (http://signal.salk.edu/tdnaprimers.2.html)
SECTION: Prepare primer working solution
2. Create a 10 mM working solution of the primer by diluting 10 µL of the stock into 90 µL of water.
SECTION: Preparing the PCR template
3. Using a 0.5 mm sampling device take a single leaf punch and add to 20 µL of dilution buffer. 
SECTION: Preparing the PCR template
4. 5 µL of DNARelease Additive
SECTION: Preparing the PCR template
5. Mash the sample with the end of a pipette tip
SECTION: Preparing the PCR template
6. Incubate sample at room temperature for 5 mins
SECTION: Preparing the PCR template
7. Incubate sample at 98 oC for 2 mins
SECTION: Preparing the PCR template
8. Spin down sample for 1 min max speed in a mini centrifuge
SECTION: Setting up the PCR reaction
9. Add the following components to a PCR tube. Set up one reaction with the diluted template from the putative T-DNA insertion line and one with WT arabidopsis as a negative control. The reaction will result in amplification if the T-DNA insert is present.

     Component      Amount        2x Phire Master Mix      10 µL        Diluted template      1 µL        LBb1.3 primer [10 mM]       1 µL        RP [10 mM]      1 µL        dH2O      7 µL   
Note: LBb1.3 (ATTTTGCCGATTTCGGAAC) is the recommended sequencing primer for SALK lines. If you have SAIL, Wisc_Lox or GABI-KAT lines you will need to use a different border primer

11.  Run the following PCR program

      Step  Temperature  Time  Cycles    Initial denaturation  98  5 min  1    Denaturation  98  5s  40    Annealing  *  5s    Extension  72  20s    Final Extension  72  1 min  1  
 
12. Analyze the resulting products by gel electrophoresis
SECTION: Setting up the PCR reaction
10. In a second PCR tube set up the following reaction. Set up one reaction for the putative T-DNA insertion line and one for WT arabidopsis as a positive control. This reaction tests for the absence of a TDNA insertion. If a T-DNA insertion is present there will be no amplification product as the distance between the L and R primers. The WT control should give a product, and heterozygous lines will also give a product as there is one WT allele.