Figure 1. Schematic representation of the Megaprimer PCR method. Step A1. The flanking regions (~1,000 bp) of the target gene are amplified by PCR (Primer pair L1 + L2 and R1 + R2). The L2 and R2 primers have extensions with homology to the antibiotic resistance cassette. Step A2. The antibiotic resistance cassette is amplified by PCR (primer pair L + R). Step B. The three PCR products of the first PCR reactions are combined. The flanking regions anneal to the antibiotic resistance cassette and one large PCR product is formed.

Materials and Reagents

1. Amplitaq DNA polymerase including 25 mM MgCl₂ and 10x reaction buffer (Applied Biosystems^®, catalog number: N8080171 )
   
2. PWO DNA polymerase including 25 mM MgSO₄ and 10x reaction buffer (Roche Diagnostics, catalog number: 11644955001 )
   
3. 10 mM dNTP mix
   
4. Sterile Milli-Q water
   
5. QIAquick PCR purification kit (QIAGEN, catalog number: 28104 )
   
6. Agarose
   
7. Ethidium bromide
   
8. 0.5x TBE
   
9. 100 bp perfect DNA ladder (Merck Millipore, catalog number: 70539 )
   
10. 1 kb perfect DNA ladder (Merck Millipore, catalog number: 70537 )
    
11. 1x PBS
    
12. Bacto-agar (BD Biosciences, catalog number: 212030 )
    
13. Bacto-Brain Heart Infusion medium (BD Biosciences, catalog number: 237500 )
    
14. Glycerol
    
15. M-IV competent non-typeable Haemophilus influenzae (Herriott et al., 1970), for protocol see below.
    

Equipment

1. T100 thermal cycler (Bio-Rad Laboratories)
   
2. Nanodrop spectrophotometer (Thermo Fisher Scientific, Nanodrop, model: ND1000 )
   
3. Centrifuge (Eppendorf, model: 5810 )
   
4. Microcentrifuge (Eppendorf, model: 5417R )
   
5. Shaker (New Brunswick Scientific, model: Innova 4000 )
   
6. 37 °C, 5% CO₂ incubator (BINDER GmbH, model: CB 150 )
   
7. DNA gel electrophoresis equipment

Procedure