Identification of a second insertion in a GABI-Kat line

It is possible to re-analyze GABI-Kat lines and to generate FSTs for a second time. The method can be applied for multiple plants in parallel or for individual plants:

Plant material and growth conditions

Arabidopsis thaliana plants of the GABI-Kat collection are grown for 4 weeks under short-day conditions (8 h light/16 h dark) and then transferred to long day (16 h light). Temperature is regulated during the light period to 22 +/- 2 °C and in the dark phase to 20 +/- 2 °C with a relative humidity of 55-60%.

Oligos

DNA preparation

Please check the DNA preparation page.

FST generation

All enzymes were obtained from New England Biolabs (Frankfurt/Main, Germany).

Digest 10-100 ng genomic DNA with BfaI by mixing 1 µL of each genomic DNA sample with 2 µL NEBuffer4, 1 µL BfaI (5 U) and 16 µL H₂O. Incubate the samples for 3-5 h at 37 °C. Inactivate the enzyme at 65 °C for 20 min.

Prepare the BfaI adapter solution by mixing 25 µL of the oligo ADP2 (100 µM), 25 µL of the oligo ADP3 (100 µM), 10 µL NEBuffer4 and 40 µL H₂O. Heat the mixture to 95 °C and let it cool down to 20 °C over a period of 2.5 h using a thermocycler. The adapter can be stored at -20 °C for several weeks.

For each ligation reaction prepare 5 µL of a ligation mixture by mixing 0.5 µL NEBuffer4, 0.2 µL ATP (100 mM), 0.625 µL BSA (1 µg/µL equivalent to “10x”), 1 µL BfaI adapter solution (see above), 0.16 µL T4 DNA Ligase (6 Weiss U/µL) and 2.515 µL H₂O.

Add 5 µL of the ligation mixture to the 20 µL of the BfaI digested genomic DNA (see above) for each sample. Incubate the samples over night at room temperature. Inactivate the ligase at 65 °C for 10 min. The samples can be stored at -20 °C for several weeks.

Mix 5 µL of each of the ligated samples (see above) with 2 µL NEBuffer2, 2 µL BSA (1 µg/µL equivalent to “10x”), 0.5 µL XhoI (10 U) and 10.5 µL H₂O. Incubate the samples for 3 h at 37 °C. Inactivate the enzyme at 65 °C for 20 min.

The XhoI digested samples are used as template in a nested PCR. In the first PCR 10 µL of the XhoI digested DNA are mixed with 5 µL Thermo Pol buffer, 1 µL dNTPs (10 mM), 2 µL AP1m primer (10 µM), 2 µL o8738 primer (10 µM), 0.2 µL Taq DNA polymerase (5 U/µL) and 29.8 µL H₂O. Amplification is performed using the following parameters: 94 °C for 2 min, 30 cycles of 94 °C for 30 sec, 60 °C for 30 sec and 72 °C for 2 min, then final elongation at 72 °C for 2 min. In the second PCR(s) 1 µL of the product of the first PCR is mixed with 5 µL Thermo Pol buffer, 1 µL dNTPs (10 mM), 4 µL of a 1:1 mixture of the primers AP2A and AP2C (both 10 µM) or 4 µL of a similar mixture of the primers AP2G and AP2T, 2 µL o8648 primer (10 µM), 0.2 µL Taq DNA polymerase (5 U/µL) and 36.8 µL H₂O. Amplification is again performed using the following parameters: 94 °C for 2 min, 30 cycles of 94 °C for 30 sec, 60 °C for 30 sec and 72 °C for 2 min, then final elongation at 72 °C for 2 min.

Purify the PCR products from the second PCRs using the EXO-SAP-it kit (USB, USA) and sequence them with the primer o8409.

Note 1: This method is used to replace the method, which is described in Strizhov et al. (2003). See reference [2] in the Publication section.
Note 2: The XhoI digestion is performed in order to prevent amplification of BfaI fragments resulting from the pAC161 plasmid only.