ࡱ> kmjO $bjbj&& .tgLegLe> III]]]8L]'^S({{{: C'E'E'E'E'E'E'$)j,ji'I"i'{{'~'"""8{I{C'"C'""O%1%{Z}H g%/''0'o%,, `,%,I%"i'i'!', : PCR Polymerases we use: OneTaq (NEB) for basic PCR and colony PCR Takara LA (Takara Bio) for making deletion constructs this enzyme is very reliable but has low fidelity. Never use for cloning. Expensive. Phusion (NEB) for cloning very high fidelity enzyme, use for cloning and transformations Q5 (NEB) for cloning very high fidelity enzyme, use for cloning and transformations, alternative to Phusion Phusion and Q5 work with identical PCR programs but have different substrate preferences. For a PCR with a new set of primers, I normally set up a PCR with each and use whichever works best Template DNA for PCR Should be very dilute use 1:10 dilutions of genomic DNA preps or cDNA, 1:500 dilutions of plasmid DNA. More DNA rarely improves yield and often inhibits the reaction Primers for PCR Oligos should be more GC rich at the 5 end, have two Gs and Cs in the 3 five nucleotides, and an A/T as the 3 nucleotide. A GC profile something like this is best, but sometimes you just have to take whatever is there  SHAPE \* MERGEFORMAT  Try to design primers with a predicted Therm. Tm of ~55( (by the NEB calculator  HYPERLINK "http://tmcalculator.neb.com/" http://tmcalculator.neb.com/ in OneTaq mode). These will reliably work with a 50( annealing temperature in a OneTaq or Takara PCR reaction for yeast targets you have to be more careful about annealing temperatures for mammalian targets, so use the Tm recommended by the calculator. Try to keep the length within 20-30 nt. If you are going to use Phusion or Q5 polymerases, design your primers in the Phusion or Q5 mode aiming at Tm 60-65(, then run the reaction at the annealing temperature the calculator recommends. On arrival, dilute primers to 100pM/(l the amount of water required for this is usually written on the tube or accompanying sheet. It is often recommended to make a further 10pM/(l working solution but in reality most people just use the 100pM/ul stock directly (but use 10x less obviously). What size of reaction do I need? Analytical PCR (is there a band or not?) 12.5(l is fine Preparative PCR (for cloning, as a probe template, etc.) 50(l is plenty to run a bit out, clean, quantify and have plenty left to work with PCR for transformation in yeast - 40(l per transformation (run out 3(l, directly transform 34(l) Master mixes When setting up multiple reactions, make a master mix. This should contain everything except the DNA template. Make enough for an extra reaction or two to allow for pipetting errors. After youve aliquoted the mix into individual reactions, add the DNA. Mixing the reactions is not necessary as heating will do this for you. OneTaq PCR OneTaq comes as a 2x pre-mix with loading dye already incorporated. It is stable for ~1 month in the fridge to avoid having to thaw aliquots. We use this enzyme for all our genotyping. 12.5(l25(l50(l2x OneTaq Hotstart6.2512.525Oligo 1 (10pM/(l)0.250.51Oligo 2 (10pM/(l)0.250.51DNA0.250.51dH2O5.51122 PCR conditions: 94( 1min 94( 15s \ *50( 15s | x 35 cycles 68( 1min/kb / 68( 5min * This annealing temperature should be calculated separately for each primer pair using the calculator here  HYPERLINK "http://tmcalculator.neb.com/%23!/main" http://tmcalculator.neb.com/#!/main. There is no need for a 4/10 hold after OneTaq PCR. Please dont as it stresses the PCR machine. Taq products sit happily at room temperature for days. Phusion PCR Normally 40 or 50(l PCRs, Phusion is a good cloning enzyme due to low error rate. *Set Phusion reactions up on ice and transfer directly to pre-heated PCR block! 40(l50(l5x Phusion buffer81010mM dNTPs0.81Oligo 1 (10pM/(l)0.81Oligo 2 (10pM/(l)0.81DNA0.81Phusion (NEB)0.40.5dH2O28.435.5 PCR conditions: 98( 30s 98( 10s \ *53( 10s | x 35 cycles 72( 15-30s/kb / 72( 5min 10( hold * This annealing temperature should be calculated separately for each primer pair using the calculator here  HYPERLINK "http://tmcalculator.neb.com/%23!/main" http://tmcalculator.neb.com/#!/main Q5 PCR Normally 40 or 50(l PCRs for cloning or transformation. Error rate similar to Phusion. Often best to try same PCR with Phusion and Q5 so one or other works *Set Q5 reactions up on ice and transfer directly to pre-heated PCR block! 40(l50(l5x Q5 buffer81010mM dNTPs0.81Oligo 1 (10pM/(l)22.5Oligo 2 (10pM/(l)22.5DNA0.81Q5 (NEB)0.40.5dH2O2632.5 PCR conditions: 98( 30s 98( 10s \ *53( 10s | x 35 cycles 72( 20-30s/kb / 72( 2min 10( hold * This annealing temperature should be calculated separately for each primer pair using the calculator here  HYPERLINK "http://tmcalculator.neb.com/%23!/main" http://tmcalculator.neb.com/#!/main Takara LA PCR Normally 40(l PCR for transformation, make sure you sequence inserts synthesised with this enzyme. This enzyme is expensive so dont waste it. 40(l10x LA PCR Buffer II buffer4Takara 2.5mM dNTPs6.4Oligo 1 (10pM/(l)0.8Oligo 2 (10pM/(l)0.8DNA0.8Takara LA Taq0.4dH2O26.8 PCR conditions: 94( 1min 98( 10s \ *48( 20s | x 35 cycles 68( 30s/kb / 72( 10min 10( hold * Annealing temperature is variable. Use 48 for the standard pFA6a primers.      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