PCR clone analysis on intact E. coli cells

1. Transfer 100 µl selective LB medium into wells of sterile microtiterplate (MTP)
2. Transfer isolated colonies with a toothpick into individual wells of MTP
3. Incubate MTP for 2 hours at 37°C
4. Add to each of the wells 100 µl of 40 % glycerol in LB
   Mix by pipetting
5. Prepare PCR mixture (amounts per sample) :
   30 µl PCR premix
   2 x 5 µl primers (10 µM)
   1/15 µl SuperTaq
6. Transfer 40 µl of PCR mixture into wells of a second MTP
7. Transfer 10 µl of culture into PCR mixture
   Cover PCR mixture with mineral oil when using the Bio-oven
8. PCR program :
   96°C - 2’
   96°C - 30’’
   50°C à 55°C - 30’’ 25 X
   72°C - 1’ - 5’
   72°C - 10’
   4°C - ¥
9. Run 1% TAE agarose gel with 10 µl of PCR products

PCR premix (per ml final PCR mixture)

• 100 µl 10 X SuperTaq buffer
• 4 x 2 µl dNTPs (100 mM)

500 µl H₂O