Amplification Of Cdna Inserts

1. Amplify the inserts of cDNA clones by PCR using primers complementary to vector sequences flanking both sides of the cDNA insert, as described previously (9). Add plasmid templates (1 to 10 ng) to 50 ^l of a PCR mixture containing 0.25 mM of each nucleotide, 0.2 ^M of each primer,

1 x Ex Taq buffer (Takara, Kyoto, Japan), and 1.25 units of Ex Taq polymerase (Takara, Kyoto, Japan). Perform the PCR as follows: at 95°C for 3 min; 35 cycles at 95°C for 30 s, 60°C for 1 min, and 72°C for 3 min; and at 72°C for 3 min.

2. Precipitate the PCR products in isopropanol and resuspend the DNA in 5 ^l of TE to a final concentration of about 2 ^g^l-1.

3. Check one aliquot of each reaction product on a 0.7% agarose gel to confirm amplification quality and quantity.

4. Add 2 ^l of 2 x polymer (Fuji Photo Film Co., Kanagawa, Japan) and 4 ^l of dimethyl sulfoxide (DMSO) (Kishida Chemical Co., Osaka, Japan) into 2 ^l of DNA solution in 96-well plates. Transfer the mixture into 384-well plates and mix at least 10 times using an automatic dispenser (model EDS-384S; Biotech Co., Ltd., Tokyo, Japan).

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