邢唷��>� 8:��7��欹�g ��bjbj*�*� .*H蚷bH蚷b�?�� ***8b$�,*�j��(��moooooo$X�!d��m��m��栨�$��[�Y�0��r!s�r!�r!��v��r!�� B�: qPCR We use two types of qPCR reaction � SYBR and probe. SYBR is a general purpose reagent that works with any primer set, probe PCRs are expensive to initially set up but allow multiplexing of multiple qPCR reactions in a single tube which substantially increases accuracy when measuring small differences eg: rDNA CNV. We normally use 10祃 reactions in 96 well plates, but have recently also started using 4祃 reactions in 384 well plates. Recipes given below are for 10祃 reactions, just scale for 4祃 reactions but tell the machine they are 10祃. We use the Maxima SYBR/probe mixes from Fermentas (now Thermo) The template DNA has a big impact on qPCR. qPCR is very sensitive to trace phenol so phenol:chloroform purified DNA should be diluted at least 10-fold. Genomic DNA samples should ideally be digested before use (with a restriction enzyme that doesn抰 cut in the amplicons!) to bring the fragment size to <10kb. This ensures that the DNA solution is homogenous and increases accuracy. Basic SYBR reaction 5祃 2x Maxima SYBR mix 0.3祃 primer mix, containing both primers at 10礛 0.7祃 water Make up enough of this mix for 10% more reactions than you need and aliquot 6祃 into each well. Dilute DNA samples 1:4 (if required), then add 4祃 DNA to each well. Each sample should be assayed in triplicate. Run using the following program: 95� 10 min 95� 15 sec \ 60� 1 min x40 Plate read / Melt curve (this is pre-defined when you set up the program) Designing primer sets for qPCR We use this site with default parameters, and normally order and test 2-3 sets of oligos. https://www.genscript.com/ssl-bin/app/primer Test them by running reactions in triplicate on 5x 10-fold serial dilutions of test DNA. Use inbuilt qPCR software (BioRad CFX on our systems) to calculate efficiency and reproducibility (as an R2 value) � efficiency should be close to 100%, R2 should be >0.985. Alternatively, design primers by standard methods given in the PCR protocol, aim for a Tm of 55-58 based on the NEB oligo calculator. Designing primer sets for probe PCR Start by testing the candidate primer set by SYBR-qPCR and only if they are excellent primers should you order the probe (probes are expensive). The probes we use are based on the TaqMan system, and have a fluorophore on one end and a quencher on the other. Common combinations: 6FAM � oligo � BHQ1 HEX � oligo � BHQ1 Basic probe PCR reaction 5祃 2x Maxima probe mix 0.3祃 each primer mix, containing both primers at 10礛 0.1祃 each probe (10礛) Water to 6 祃 Make up enough of this mix for 10% more reactions than you need and aliquot 6祃 into each well. Dilute DNA samples 1:4 (if required), then add 4祃 DNA to each well. Each sample should be assayed in triplicate. 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