ࡱ> 8:7g bjbj** .*HibHib? ***8b$,*j(moooooo$X!dmm$[Y0r!sr!r!vr! 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 10l reactions in 96 well plates, but have recently also started using 4l reactions in 384 well plates. Recipes given below are for 10l reactions, just scale for 4l reactions but tell the machine they are 10l. 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 doesnt 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 5l 2x Maxima SYBR mix 0.3l primer mix, containing both primers at 10M 0.7l water Make up enough of this mix for 10% more reactions than you need and aliquot 6l into each well. Dilute DNA samples 1:4 (if required), then add 4l 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 5l 2x Maxima probe mix 0.3l each primer mix, containing both primers at 10M 0.1l each probe (10M) Water to 6 l Make up enough of this mix for 10% more reactions than you need and aliquot 6l into each well. Dilute DNA samples 1:4 (if required), then add 4l DNA to each well. Each sample should be assayed in triplicate. Run using the following program: 95 10 min 95 15 sec \ 60 30 sec x40 Plate read / Melt curve (this is pre-defined when you set up the program) Yeast Genomic DNA preparation for qPCR Re-suspend 1 large colony or cells from ~100l saturated culture in 200(l Fast Lysis buffer (should be 5x107 cells strictly) Add 20-50(l glass beads and 200(l phenol/chloroform Vortex for 5min at 4(C or room temperature Spin for 5min at top speed Extract 150l of the upper phase, add 15l 3M NaOAc pH5.2 and 450l ethanol Vortex, then spin 15min at top speed Wash pellet with 1ml 70% ethanol Dry pellet 5min at RT Add 100l EB (10mM Tris pH8.0, 1mM EDTA), vortex Digest 50l gDNA 10l CutSMART 0.2l (4U) EcoRI (for example) 0.2l 1mg/ml RNase A in 100l 37 for 1 hour, then purify through a QIAQuick PCR cleanup column into 120l Load 4l per well in 10l qPCR Fast Lysis Buffer: 2% Triton-X100 1% SDS 100mM NaCl 1mM EDTA 10mM Tris pH8      FILENAME qPCR v1.1 Houseley lab  PAGE 3 A B C ) * i    E F K L J L M T U W X e f >j/0_`sǿүүүүүҫҫүΣΣΣΛΔΏΏ΋Ά h.55hV5 hz H* hz hz hz hz 5hz OJQJh hy0OJQJhz h)5hz hy05 h)hy0hz hy0 h)h)hMh)hMh)5hM5CJ(aJ(h)5CJ(aJ(2B C ) * i j  H T ' ( I J U c u gd)$a$gd) =>kls!"78L_`yzPQgd gd E88`oy{|=>P`afgnoԺԶ詶 jmh{_)h{_)OJQJhk h{_)5h{_)h{_)5h{_)h h OJQJ h h h OJQJh h E8OJQJhz h E85 h E85h E8h.5 h.55 hz 5;!^_`;<gh/0gd{_)gd E8gd &'PQklmwx )*ļjh(0JUh%hV5hLmHnHuh(jh(Uh|,jh|,U h.5h{_) h{_)h{_)h dh{_)OJQJ jh{_) jmh{_)h{_) hkh{_) hkH*hk60abt12T^lxgd{_) h.5h{_)h|,h(jh(0JU*hV50JmHnHu*jh(0JU h(0J,1h. 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