Week 5- PCR Troubles

This week has been a week of learning with some frustrations.  We have been trying to run PCR on two of our samples and we jsut can't seem to get the results we are looking for.  The first time ran a the PCR we had no aplification and the second time we ran it the positive didn't amplify.  The third time we ran the same PCR we had more amplification but some of the 12 C labels didn't amplify and they are supposed to always amplify.  This was concerning because it could have meant that we didn't have any DNA after cleaning the SIP.  So I ran a gel to be sure that there was DNA in the samples I cleaned with Tris/EDTA and sure enough there was.  This limited the mistake to the running of the PCR because with DNA in the cleaned SIP this that we successfully pulled out the 12 and 13 C bands.  This also means that we cleaned the DNA correctly as well.  We didn't contaminate the PCR because then everything would have amplified and it did not. 

We decided that it was possible that because I had not made a master mix for the PCR set up but had individually added the primers and water to the PCR tubes that I might not have gotten the solution mix up enough.  Instead, this time I made a master mix by multiplying the number of tubes I needed to PCR by the uL needed of each product in a single tube.  I mixed it all in one ependorff tube and then added the required amount to each small PCR tube.  This way the solution is thoroughly mixed before it is put into the PCR machine. 

My goals are to get the PCR to work on my first two samples so that I can continue to run PCR on the rest of the samples from Astoria.  When I do this I will be able to proceed with my project by cleaning the PCR and starting mass cloning.  This has been my first real problem during my internship and I am learning how to troubleshoot in order to find an accurate answer.