Detection and Cloning of Spliced Transcripts by RT-PCR

Materials and Reagents

I.   RT-PCR

1. QuantiTect Probe RT-PCR Kit (QIAGEN, catalog number: 204443 )
   
2. Primers (sense and antisense) (Biomers)
   
3. Isolated RNA
   
4. PCR tubes (Starlab)
   
5. Nuclease-free water (B Braun)

II.  Cloning

1. Agarose (Roth, catalog number: 2267.4 )
   
2. Ethidium bromide (AppliChem)
   
3. DNA ladders (1 kb and 100 bp) (Life Technologies, Invitrogen^TM)
4. Geneclean III Kit (Qbiogene, catalog number: 1001-600 )
   
5. Example of an acceptor plasmid: pcDNA3.1 (Life Technologies, Invitrogen^TM, catalog number: V790-20 )
   
6. Restriction endonucleases with reaction buffers (New England Biolabs)
   
7. Antarctic phosphatase with reaction buffer (New England Biolabs, catalog number: M0289L )
   
8. TaKaRa-DNA-Ligation-Kit version 2.1 (TaKaRa, catalog number: 6022 )
   
9. Ultracompetent XL2-blue bacteria (Stratagene, catalog number: 200150 )
   
10. Carbenicillin (AppliChem, catalog number: A1491 )
    
11. Bacteria cell culture tubes (Sarstedt)
    
12. Plasmid preparation Kits (QIAGEN, catalog number: 27104 , 12362 )
    
13. Tris-HCl (pH 7.8)
    
14. Sodium acetate
    
15. EDTA
    
16. NaCl
    
17. KCl
    
18. MgCl₂
    
19. MgSO₄
    
20. LB medium (see Recipes)
    
21. TAE buffer (see Recipes)
    
22. 6x loading dye (see Recipes)
    
23. SOC medium (see Recipes)
    
24. LB agar (see Recipes)
    

Equipment

1. PCR cycler (Mastercycler gradient 5331, Eppendorf, catalog number: 5331000.010 )
   
2. Microwave oven (Alaska, model: M 1001 )
   
3. Gel electrophoresis chamber (Perfect Blue electrophoresis system 40-1214, PeqLab) and power supply (Power supply E844, Consort)
   
4. UV light box (UV-Transilluminator) (Vilber)
   
5. Incubators (Thermomixer comfort, Eppendorf, catalog number: 5355000.011 ; 37 °C shaker for bacterial cell cultures Novotron, Infors HT)
   
6. Scalpel (B Braun)
   
7. Scalpel (B Braun)
   
8. Bacteria cell culture tubes (Sarstedt)

Procedure