Molecular Methods in Developmental Biology Molecular Methods in ...

46 Steinbach and Rupptionation by gel electrophoresis, specific PCR products can be identified byethidium bromide staining. For accurate quantitation, however, we recommendradiolabeling the PCR products directly during synthesis by including traceamounts of [α- 32 P]–dCTP in the PCR mix.3.3.1. Standard Single Primer Pair or Multiplex PCR Reaction1. Prepare a PCR mastermix (with a few extra reactions worth) on ice, whichconsists of the following components per reaction (the reaction may bedownscaled twofold):dH 2 O 39.9 µL10X PCR Buffer 4.8 µL10 mM dNTP 1 µLPrimer mix—gene 1 2 µL(forward and reverse primer, 25 µM each)5 U/µL Taq polymerase 0.2 µL10 µCi/µL [α- 32 P]–dCTP (3000 Ci/mmol) 0.1 µLTotal 48 µLAdditional primer pairs for multiplex PCR are accommodated by reducing theamount of dH 2 O. Mix gently, centrifuge briefly, and store on ice.2. Aliquot premix portions (48 µL or less, see item 7) into 0.5-mL PCR tubes.3. Set up an extra reaction and add 2 µL dH 2 O (the so-called minus template[–template] control; see Note 3).4. Unless your thermal cycler is equipped with a heated lid, overlay each samplewith two drops (approximately 50 µL) of light mineral oil.5. Add 2 µL of RT reaction per PCR tube. Make sure you penetrate the mineral oillayer. Mix by pipeting up and down. Centrifuge briefly.6. Place the reaction tubes in the thermal cycler and start the required PCR program.7. Because of differences in template abundance, separate primer pairs may requiredifferent cycle numbers in multiplex PCR. In that case, primers for themore abundant mRNA are added to the PCR samples after n–x cycle numbers(n is the cycle number for the less abundant mRNA; x: cycle number for themore abundant mRNA). PCR reactions are cooled to 4°C after n–x cycles,and 2 µL of primer mix for each additional gene is added (final volume: 50µL). Be sure to penetrate the mineral oil layer! Continue with the program forx cycles.8. PCR products are stable and can be stored at room temperature or 4°C until furtheranalysis.3.3.2. Size Fractionation of PCR Products by PAGEAlthough itself semiquantitative, polyacrylamide gel electrophoresis(PAGE) offers an inexpensive and convenient method for post-PCR analysis,in particular with large sample numbers. It is used to separate specific PCR