Whole Cell PCR

From: Lisa Kreppel, Ph.D. National Institutes of Health

Some critical parameters

  • Cells must be mostly alive! I pick my blast-R clones into 96 well plates and try to screen as soon as some of the wells are confluent... don't wait too long or else most cells will be dead and you will NOT get any PCR products! If you suspect that your cells are mostly dead then replica plate into a new 96 well plate.

  • LONG primers!!! I tend to go with 30mers. I shoot for Tm<50*C and try to match the primers Tm values. I don't worry about %G/C. I DO NOT use the very old fashion A:T= 2*C/G:C=4*C rule to calculate Tm! Instead I use MacVector (test PCR primer pair option) to see what the Tm is, then I drop down 3-5*C from there for the annealing temp in the PCR reaction. I do NOT use Mac Vector to pick my primers the dicty genome is too AT rich for this program to work very well. Instead you pick and run them through the program.

    Note: Macvector will often give you all kinds of warnings about primer dimers, primers that anneal within the product etc. Look with your eyeballs see if you can see it if not don't worry! The parameters of the program are quite stringent. If you don't have access to MacVector this website: http://www.appliedbiosystems.com/support/techtools/calc/ has a Tm calculator that works very well! (I just leave the salt and primer conc. set to the default setting and then drop 3-5*C for annealing temp.)

  • TEST your primer pair using your K/O plasmid and genomic dna as K/O and WT controls. Be sure to use only a tiny teeny amount of plasmid.... you want to try a mimic the conditions of the actual PCR. Sometimes I will include a mix of about 50ul of dicty culture (spin down and remove supernate) to which I add a tiny amount of my K/O plasmid to see if I can pick up both products in a single rxn.

  • The polymerase you use appears to be absolutely critical! We have had great success with two different products. HotTub by Amersham (nice but very $$$$ here) and ThermoPrime by AB Gene (they have a version sold with something called reddy mix that is very nice, has the loading dye right in it. Saves LOTs of pipetting when loading gels and makes it easier to see which wells you've added rxn mix to!). The HotTub literature suggests using it at lower elongation temps (65*C). I do the same for the Thermo Prime even though they don't say anything about using it at lower temps.

  • LONG elongation times and LOW elongation temps. A sample program:
    • 94*C 5 min
    • 94*C 30 sec <------
    • 55*C 1 min | repeat 35 X
    • 65*C 3-4 min ------
    • 65*C 10 min

The BASIC Protocol

  1. Into a 96 well PCR plate I put 75ul of culture from each well of my 96 well clone plate. Be sure to pipette up and down to resuspend the cells before removing your sample for PCR. Obviously you must change tips between each well.
  2. Leave 1-2 wells empty for negative no template controls and if you're worried leave another empty to be used for a positive control using your K/O plasmid.
  3. Spin down the PCR plate to pellet cells.
  4. Then CAREFULLY aspirate off the media. I use a yellow tip on the end of a pasture pipette. CHANGE TIPS between each well. It's frighteningly easy to cross contaminate at this stage! It's not critical to remove every last bit of media. A few remaining ul are not a problem.
  5. Put plate on ice.
  6. Mix up your PCR rxn mix as a big cocktail. I find I have to divide it into two eppendorf tubes when I have a full 96 well plate to screen. Also you will need to mix up enough for 1 extra rxn for every 10 rxns you want to do (i.e.: mix up at least enough for 110 rxns if you have 100 rxns to perform). I go for small 25 ul reactions to save reagents. Sounds too obvious but it's critical that you mix the cocktail really well! It's easy to not get full mixing when you have nearly a ml in the tube.Here's a sample cocktail for 1 sample:
    • 2.5 ul 10X PCR buffer
    • 0.25 ul 25mM dNTPs
    • 0.75 ul 6uM primer A
    • 0.75 ul 6uM primer B
    • 19.5 ul dH2O
    • 0.25 ul Polymerase (see above for brand)
  7. Use 24 ul per rxn (I assume that at least 1 ul of media is left behind in wells) place cocktail into very bottom of well and CHANGE tips between each sample!!! I don't make any special effort to resuspend the cell pellet, just put the cocktail right on top of the cells. as you can see I have my dNTPs and primers at pretty high concentrations.... this is not necessary, I just find it convenient for my lab uses. You will also notice that this protocol is very pipette tip intensive.... I really think this is important, every time I try to save tips I have cross contamination... so I just use lots-o-tips.