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2. Reaction Mix (per assay):
Note: Use sterile, silicanized microfuge tubes.
This is the standard protocol for the control template.
order of Final
Component Addition Volume Concentration
Double-distilled, 1 53.5 ul
Sterile H20
CLOX] Reaction Buffer 2 10 ul [lX]
dNTPs Mix, 1.25 mM 3 16 ul 200 uM (in
(in each DNTP) each dNTP)
Control Primer #1 4 5 ul 1.0 um
Control Primer #2 5 5 ul 1.0 um
Control Template 6 10 ul 1 ng/assay
(10-1 dilut.)
Tag Polymerass 7 0.5 ul 2.5 Units/
asssay
Overlay the samples (100 ul in 0.5 ml capped polypropylene tubes) with 100 ul of mineral oil (such as Sigma Cat. No. 400-5 or M-3516), to prevent evaporation. The oil layer will not interfere when withdrawing aliquots from the sample for analysis. If the whole sample (100 ul) needs to be recovered, extract the sample with 100 ul of chloroform (high purity grade) . The aqueous phase, containing the sample, will form a micelle near the meniscus. It will then be easier to collect the sample by withdrawing it with an Eppendorf pipette.
Larger master mixes can be prepared and aliquoted before adding the template and the T-ag Polymerase. If you suspect that your template is contaminated with proteases (as may be the case with genomic DNA), add the Taa Polymerase after the initial denaturation step. The high temperature incubation should inactivate proteases and prevent degradation of the Taa Polymerase.
3. Experimental Protocol:
Note: Performance of the PCR technique with the GeneAmpTM DNA Amplification Reagent Xit, including Taq Polymerase, can be automated with the PERKIN ELMER CETUS DNA Thermal
Cycler. The instrument enhances the user's ability to control and optimize the PCR technique to achieve maximum amplification efficiency. Operating instructions and protocol recommendations f or use with the DNA Thermal Cycler are contained in the Operator Manual. The f ollowing manual procedure is recommended for those using heat blocks or water baths.
Use heat blocks or water baths (one each) adjusted to the following temperatures: 370C, 720C, 940C.
Initial template denaturation step: 1 min. 30 sec. at 940C. Afterwards, a typical cycle profile will be:
" minutes at 370C (annealing)
" minutes at 720C (extension)
" minute at 940C (denaturation)
Repeat for a total of 25 cycles. At the end of the 25th cycle, OMIT the heat denaturation step and extend the extension step by an additional 7 minutes. Following the termination of the assay, let the samples come back down to room temperature. The samples can be kept at +40C overnight, pending further analysis.
Note: A. The size of the target sequence in your sample DNA will directly impact the minimum time required for proper
extension (720C incubation step) . An optimization of the temperature cycling profile should be performed for each individual template, in order to obtain maximum amplification efficiency.
B. Be aware that there is a significant lag time (30 seconds to 1 minute) between the time tubes are transferred from one temperature to another and the time the samples actually reach the new set temperature. The lag time will be a function of the type of heat source and tubes used, the sample volume and the temperature
differential between incubation steps. Adjust the
length of the incubation steps to compensate for this
lag time.
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