WPA Galliformes Genetics Group

There are several primary reasons why most people, particularly those new to the lab, have problems with PCR.  I will try and list some of those problems and potential solutions to try first:


· Non-amplification of template

· One of the reagents was left out of the mixture:
Although this sounds pretty goofy, it actually happens relatively frequently, especially for folks new to PCR.  In this case, setting up your cocktail and running the reaction again should ease your troubled mind.  One thing that folks in our lab do, at least initially, is to check off each reagent from the list of those in the cocktail to ensure that everything gets in there.  Another potential solution (because Taq is often the reagent left out) is to use Taq with an inert red dye to indicate its addition (this is available from AbGene or Sigma, among others.
· Pipetting error:
Commonly, when you are preparing the PCR cocktail for thermal cycling, you may be preparing it for very few samples.  As is always the case, you want to try and make your cocktail for all of the samples and then pipet the neccessary reaction volume into your PCR tubes/plates.  Similarly, if you are using very low reaction volumes (5ul or 10ul) you may want to step the volume up to 20ul or 50ul to see if that helps the problem.  You can always drop back down later.
· Poor template mixing:
This is troublesome because it often happens.  Template DNA solutions that have been frozen or in the fridge for awhile are particularly prone to this problem.  The solution is to mix the template well (using vortex mixer or sqlooshing with your pipet) to ensure the template is well-mixed before adding it to the reaction.  The same goes for PCR product being prepared for a sequencing/sizing run.
· Inhibition of PCR reaction:
This one is a real doozy.  We have had this occur in our lab numerous times.  In most cases, we have identified the problem as arising from bad ddH20 that contained some inhibitory reagent/fungus/something else.  Making aliquots of ddH20 and keeping them in the fridge for single-use can help.  If this does not help, we typically don't mess around with trying different reagents for too long.  We will usually try new reagent aliquots (aliquot everything!) to see if that fixes the problem.  If it does, awesome.  If it does not, you may need to buy new reagents to see if you can get those to work.  Once you have things working again, you can go back to experiment and see what is causing the problem.  Typically, this is very time consuming and frustrating, and we often do not bother.  However, if the problem is common, it may help to identify the individual component that is the culprit.

· Poor Template Amplification

· Too Few Cycles
Stepping up the cycle number will greatly increase your amount of product (it will usually [u]not[u] increase its quality once you get visible product, however).  In fact, too few cycles may result in PCR product that is not visible (particularly in agarose gels stained with EtBR) leading you to believe that you have not amplified anything.
· Stuttery/Smeared Bands
There are several potential causes here. 

· The first one to be sure of is that you have kept your reaction mix on ice while making it - particularly if you are not using heat-activated taq polymerase.  If not on ice, the reaction can begin before you get the tubes in the cycler - which decreases the specificity of your primers allowing them to bind in numerous places leading to production of bands with varying sizes that look stuttery or smeared.
· You can also have problems with your annealing temperatures.  Similar to the situation detailed above, low annealing temperatures decrease the specificity of your primer when it anneals to the DNA you are attempting to amplify.  Higher annealing temperatures increase this specificity.  However, annealing temperatures that are too high may (1) melt your primer, and (2) result in no amplification of PCR product.  Therefore, one of the better ways to test your annealing temperatures is to use a gradient thermocycler which will allow you to run a progression of temperatures across the block so you can test many temps. at once.  Remember, that if you use someone else's thermocycler with a gradient option, the temperatures that will work in your lab may be somewhat different than those that worked elsewhere.  So, be forewarned




More to come as i have time...