Methods of molecular identifications and lab. protocols
UP-PCR. S. Bulat, modified version by M. Lübeck. UP-PCR was initially developed using a high ramping thermo-cycler TC-1000M (PNPI, St. Petersburg, Russia) for 30 cycles along with 0.5 ml narrow but thick-walled tubes (Lenmedpolimer, St.Petersburg, Russia). The rate of ramping between the different temperatures in this thermo-cycler is about 4oC/s, which is higher than the ramping rate of the most commonly used thermo-cyclers. By optimization, however, I have found that it is possible to obtain exactly the same banding profiles using standard 0.5 ml tubes or 0.2 ml and either the MJ Research thermal cycler (Minicycler, Model PTC-150, ramping rate 2.4oC/s), or the Eppendorf Personal Cycler. Also I have found that identical profiles can be obtained using different polymerases (e.g. T.sp. polymerase or Dynazyme) together with buffers with different composition (Lübeck et al., 1999). Dynazyme (Finnzymes OY, Espoo, Finland) and the buffer supplied with the enzyme are used in the protocol below. The concentration of target DNA for optimal PCR reactions should be adjusted by running PCR using 2 or 3 different DNA concentrations from each sample. Make 10 and 100 fold dilution of the DNA sample. Too much template DNA often inhibits proper PCR reactions. Each time a new batch of polymerase, primers or dNTP are used, it is recommended to test them in PCR by making dilution series. In order to minimize the risk of contamination and facilitate comparison of the different reactions in each PCR series, the amount of pipetting is kept to a minimum. All common reagents that should be included in each reaction (listed below) are added to one microfuge tube and subsequently distributed into the different PCR tubes. E.g. you would like to amplify ITS1 from 4 isolates and test their DNA using three concentrations. Then the same reagents should be used for each reaction except for the DNA. Therefore all reagents multiplied by 12 (except the DNA) are added to one microfuge tube and subsequently distributed into 12 labeled PCR tubes. Then the different DNA can be separately added to the individual (corresponding) tubes. Preparation of aliquots of dNTPs: Prepare 20 µl aliquots of 10 mM dNTP (10 mM each of dATP, dCTP, dGTP, dTTP) stock solutions. Store the aliquots at -20oC and the stock solutions at - 80oC. Preparation of primers: Prepare 100 pmol/ l of each primer and test optimal primer amount in PCR. Final amount should be between 10 and 100 pmol in each reaction. Preparation of 1 ml 1 X Dynazyme buffer, mix: 10 X Dynazyme buffer is supplied with the polymerase. Store the Dynazyme buffer at +4oC, because of its content of 15 mM MgCl2. Repeated freezing and thawing of MgCl2 can result in the precipitation or accumulation of MgCl2 in an insoluble form. For UP-PCR the exact MgCl2 concentration is important for the efficiency of the reaction. Therefore it is recommended to make a 100 mM MgCl2- stock solution which should be stored at +4oC. 100 µl 10 X buffer, 20 µl 100 mM MgCl2, 20 µl 10 mM dNTP and 860 µl H2O (sterile). 1 X buffer containing dNTP can be stored at +4oC (1 - 2 weeks). For one ?20? µl PCR reaction, mix: 1 µl DNA 0.1-1 µl primer, depending on experimental results (see ?preparation of primers?) 0.2-0.3 µl Dynazyme (2U/µl) 18 µl 1 X buffer. Note that the final amount may exceed 20 µl. PCR in Eppendorf Personal Cycler: First cycle: 1. initial denaturation at 94oC for 2 min. 2. annealing at 53-56oC for 40 sec. (annealing temp. depending on the primer) 3. extension at 72oC for 30 sec. Followed by 29 cycles: 1. denaturation at 92oC for 20 sec. 2. annealing at 53-56oC for 40 sec. (annealing temp. depending on the primer) 3. extension at 72oC for 30 sec. Last cycle: The extension step should be prolonged to 2 min.
posted by Mette Lübeck on 04 Oct, 2004