Materials and Reagents

1. Restriction of genomic DNA using endonucleases
   1. Genomic DNAs from the samples to be analysed at concentrations of 100 ng/µl.
      
   2. Endonuclease enzyme insensitive to DNA methylation, generating cohesive ends
      Note: TE sequence ends have to be devoid of any corresponding restriction sites.
      
      
2. Adapter ligation
   3. Two oligonucleotides which form a double-stranded adapter, with an overhang complementary to the overhang left by the restriction enzyme used. In the case of EcoRI (Thermo Fisher Scientific, Fermentas) the 5’ overhang is AATT and the oligonucleotide sequences are 5’-CTCGTAGACTGCGTACC-3’ and 5’-AATTGGTACGCAGTCTAC-3’. Annealing of the two oligonucleotides will constitute the adapter (purple rectangles in Figure 1).
      
   4. T4 DNA ligase (1-3 U/μl) with the corresponding ligase buffer (Promega Corporation, catalog number: M1801 )
      
   5. Sterile ultrapure water
      
      
3. Pre-amplification
   6. PCR reagents
      1. Homemade Taq DNA polymerase is of sufficient quality for the PCR reactions but Taq Promega has also been used (Promega corporation, catalog number: M8301 ) with the corresponding Taq polymerase buffer.
         
      2. 25 mM MgCl₂
         
      3. dNTPs (10 µM each)
         
   7. Pre-amplification primers (10 μM each)
      1. One primer is anchored in the adapter: 5’-GACTGCGTACCAATTC-3’ (primer P1 in Figure 1).
         
      2. The other primer is anchored in the TE of interest, close to its end to limit the size of amplicon (primer P2 in Figure 1).
         
         
4. Selective amplification
   8. PCR reagents (see pre-amplification step)
      
   9. Selective amplification primers (10 μM each)
      
      1. One primer is anchored in the adapter, with the addition of 3 nucleotides at the 3’ end (primer P3 in Figure 1) to limit the number of bands to be visualized and analyzed by gel electrophoresis. Several combinations of A, T, G and C should be used to target different genomic localizations of TE insertion sites.
         
      2. The other primer is defined to perform a nested PCR in comparison with the pre-amplification step (primer P4 in Figure 1); this primer is labelled with a 5’-IRDye. DNA labelled with IRDye (infrared dye) has to be stored in the dark at -20 °C. To minimize exposure to light, wrap the tube in aluminium foil.
         
         
5. Acrylamide gel for SSAP profile analysis
   10. Kimwipes
       
   11. Urea (Merck KGaA, catalog number: 1.08488.1000 )
       
   12. Long Ranger Acrylamide (50%) (Lonza, catalog number: 50611 )
       
   13. APS (Bio-Rad Laboratories, catalog number: 161-0700 )
       
   14. Temed (Bio-Rad Laboratories, catalog number: 161-0801 )
       
   15. Absolute ethanol
       
   16. Formamide (Sigma-Aldrich, catalog number: F9037 )
       
   17. 10x TBE buffer (Tris/Borate/EDTA) (see Recipes)
       
   18. 5.5% acrylamide gel (see Recipes)
       
   19. Loading buffer (see Recipes)
       

Equipment

1. 1.5 ml Eppendorf tubes
   
2. Centrifuge
   
3. Parafilm
   
4. Incubator or oven
   
5. Water bath
   
6. 96-well PCR plates
   
7. 0.45 μm filters
   
8. LI-COR DNA analyzer (LI-COR) and corresponding equipment (plates, combs, spacers...)
   
9. Thermal cycler
   
10. Horizontal shaker
    

Procedure