Hello Everyone,
I need an efficient protocol to genotyoping my Arabidopsis mutants obtained from "SAIL" and "SALK" seed-stocks. I'm screening homozygous plants for genetic transformation and functional complementation. Any suggestion, from primers' desiging to experimental procedures to collect and genotyping the plants, will be very appreciated. Thanks so much!!
You can use this primer design tool at T-DNA express. There you can also find information on how to set up PCR and which T-DNA border primers to use. http://signal.salk.edu/tdnaprimers.2.html
Below a simple and fast DNA extraction protocol which usually works fine
Miniprep of genomic DNA from plant material
1. Put a small leaf into a 1,5 ml eppendorf tube, freeze in -20°C (or freeze in liquid N2 and store at -80°C for later use)
2. Add 750 µl EDM-buffer and incubate at 95°C for 5 minutes
3. Leave in room temperature over night
4. Add 750 µl of isopropanol and mix
5. Centrifuge at full speed for 5 minutes
6. Remove the supernatant
7. Wash with 1 ml 70% EtOH, invert several times and centrifuge full speed 2 minutes
8. Remove the supernatant
9. Dry at 37°C or RT
10. Resuspend in 150 µl TE-buffer (or H20). Let shake for approximately 10 minutes.
11. Centrifuge 1 minute before use as PCR template
EDM-buffer: 200mM Triss-HCl, 250 mM NaCl, 25 mM EDTA, 0,5% SDS
I always used the Neff method for DNA isolation, which is very similar to the protocol from Anders, but after grinding the leaves and adding the buffer you can directly proceed with DNA precipitation. With plant PCR it is important to use only a small amount of your preparation (1-2%) since contaminants hamper the PCR reaction.
The primer design from the Signal T-DNA tool indeed works fine. Otherwise, blast the flanking sequence to genomic DNA and design the primers based on the T-DNA position using appropriate tool (such as Vector NTI). Until now for only 5% of the insertion lines (>40 analysed) I had to redesign the primers.
For such genotyping I often perform "leaf PCR" in which a small piece of leaf or cotyledon tissue is added directly to the PCR reaction, similar to the concept of "colony PCR".
Thanks Doctors Nilsson, Graaff and Zwack, I will try the suggested protocols.
Best Regards !!!
After the successful primer designing from the T-DNA express, you can also use the one-step DNA extraction using Edward's solution and use 1ul crushed sample for the PCR reaction which might be an easy way for genotyping.
Please find the attached protocol: One-step DNA extraction from Arabidopsis thaliana
https://www.arabidopsis.org/download_files/Protocols/Protocol_Kasajima.pdf
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