2. For one 96-well PCR prepare a total of 3.19 ml reaction mix (enough for 110 reactions) containing 1X PCR buffer (supplied with DyNAzyme EXT DNA Polymerase-MJ Research), 200 µM dNTPs, 0.3 µM primers and 22 U DNA polymerase.
3. Add 29 µl of the mix to each well using a multichannel pipette.
4. Add 1 µl 1:100 cell dilution into each well using a multichannel pipette, mix thoroughly and centrifuge at 4000 rpm for one minute.
5. Place plate in PCR machine.
Cycling Conditions for amplification of clones in pFLC-I:
94 ° C 1min__________
94 ° C 30sec
66° -2 ℃ 45sec 5 Cycles
72 ℃ 3min+12sec_____
94 ° C 30sec
56 ° C 45sec 30 Cycles
72 ℃ 3min+6sec______
72 ℃ 15min__________
4 ℃ ∞
Cycling Conditions for amplification of clones in pOT2A:
94 ° C 1min__________
94 ° C 30sec
67 °-1 ℃ 45sec 5 Cycles
68 ℃ 3min _____
94 ° C 1 min
62 ° C 45sec 32 Cycles
68 ° C 4min+6sec______
68 ° C 10min__________
4 ℃ ∞
Cycling Conditions for amplification of clones in pBS:
95 ° C 1min__________
95 ℃ 30sec
70 °-2 ℃ 45sec 5 Cycles
72 ℃ 3min+12sec_____
95 ℃ 30sec
60 ℃ 45sec 28 Cycles
72 ℃ 3min+6sec______
72 ℃ 15min__________
4 ° C ∞
6. To monitor the PCR reaction, samples are quantitated and sized by electrophoresis through 1% agarose in TAE (40 mM Tris, 20 mM acetate, 2mM EDTA, pH 8.1) using either the centipede (96 samples) or millipede (192 samples) electrophoresis apparatus (Owl Scientific).
7. The samples are now ready for G-50 purification.
