Pcr

1. The reagents for each 25 |L Tay Sachs ASA assay reaction are as follows:

0.05 |L each dNTP (dATP, dCTP, dTTP, dGTP), 100 mM concentrations

(200 |M dNTPs final concentration) 0.08 |L primer 2659U29M, 50 pmoles/|L ( 4 pmoles total/ 25 |L reaction) 0.06 |L primer 2659U30N, 50 pmoles/|L ( 3 pmoles total/ 25 |L reaction) 0.08 |L primer 2729L26, 50 pmoles/|L ( 4 pmoles total/ 25 |L reaction) 0.12 |L primer 2915L24M, 50 pmoles/|L ( 6 pmoles total/ 25 |L reaction) 0.12 |L primer 2915L24N, 50 pmoles/|L ( 6 pmoles total/ 25 |L reaction) 0.12 |L primer 2779U25, 50 pmoles/|L ( 6 pmoles total/ 25 |L reaction) 0.20 |L primer 1726L30M, 50 pmoles/|L (10 pmoles total/ 25 |L reaction) 0.14 |L primer 1726L30NA, 50 pmoles/|L ( 7 pmoles total/ 25 |L reaction) 0.10 |L primer 1660L25MA, 50 pmoles/|L ( 5 pmoles total/ 25 |L reaction) 0.14 |L primer 1660L27N , 50 pmoles/|L ( 7 pmoles total/ 25 |L reaction) 0.36 | L primer 1666L31M, 50 pmoles/| L (18 pmoles total/ 25 | L reaction) 0.20 | L primer 1666L31NA, 50 pmoles/| L (10 pmoles total/ 25 | L reaction) 0.24 |L primer 1559U29B, 50 pmoles/|L (12 pmoles total/ 25 |L reaction) 0.4 |L Taq polymerase (5 U/|L)

2.0 |L 100 ng genomic DNA plus ddH2O to bring volume to 2.0 |L 25.0 |L total

2. We typically prepare a large reaction kit that will test 100 samples to reduce the set up time needed for each reaction (see Note 6). The kit is prepared as shown in Table 2, and includes ddH2O, buffer, dNTPs, and all primers. The kits are prepared and stored in aliquots of approx 40 single reactions at -20°C in a non-frost free freezer-protected from light. When a test is required, an appropriate number of aliquots are thawed, and in each PCR tube the following is prepared:

22.6 |L Tay Sachs ASA kit 0.4 | L Taq polymerase

2.0 |L 100 ng genomic DNA plus ddH2O to bring volume to 2.0 |L

3. For each PCR reaction, appropriate control samples should be prepared. Controls required include a normal sample, positive controls for each mutation tested (see Note 7) as well as a negative control containing all reagents but no DNA.

Table 2

Preparation of PCR Kit for Tay Sachs ASA Reactions"

Reagents for 100 Tay Sachs ASA reactions

DdH2O

10X PCR buffer, containing 1.5 mM MgCl2 dNTPs: use 100 mM stocks of dNTPs

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