PCR+Troubleshooting

=Last week we attempted to do PCRs. In hand, we already had alot of PCR buffer, dNTPs, primers and plastic tubes from last school year. In September, we ordered only taq polymerase, DNA Template, linearized vector, ligase enzyme and bacterial cells from Genotyp company.=


 * 1) = Because the taq polymerase has been problematic in the past, we tried different aliquots in our reactions. We first diluted the stock enzyme five times (i.e., 2ul enzyme + 8 uL water). We used 1, 2, and 3 microliters of the diluted enzyme. All other reagents were used in the preset aliquots instructed in the lab manual. Using gel electrophoresis (Genetics lab in Jordan Lab), we checked the PCRs. None of the reactions worked. =
 * 2) = We next suspected that the DNA template (recent shipment) was not good. Using a nanodropper (Dr. Severson's lab in ND Biology Dept), we measured the concentration of the DNA template (2 tubes that were shipped to us), last year's DNA template, the forward and reverse primers, and the linearized vector. The nanodropper measures the absorbance at a certain wavelength for nucleic acids (i.e., 260nm). A "blank" is required, which is normally what the nuclei acid is diluted in. We used water as our blank. =
 * == DNA Template (both tubes shipped to us this month) had no DNA. This is the main reason why the PCRs didn't work. ==
 * == Last year's DNA template had some DNA, but not a high amount. Around 6-7 nanograms per microliter. ==
 * == Forward and Reverse primers were about 15-17 ng/ul each. ==
 * == Linearized vector (this shipment) had about 6-7 ng/ul. ==
 * == Our positive control (a plasmid from Francis' lab) was ~186 ng/ul. This was a very good concentration, and we would of liked to see something close to this value in the GENO reagents. ==
 * 1) = We then tried to redo the PCRs with last year's DNA template and different taq polmerases. Aprell ran the reactions on a gel in her lab. =
 * == PCR #1: Genotyp buffer, primers, and dNTPs as well as Platinum Taq from Invitrogen Company. This reaction produced a PCR product at ~1Kb (right size), but the product was not strong. __However, it was good enough to continue cloning. This PCR was chosen for the next step, ligation.__ ==
 * == PCR #2: Genotyp primers only and GoTo Taq Mix from Promega Company. This specialized taq mix already includes buffers, dNTPs and Taq polymerase. This reaction produced a PCR product at ~1Kb (right size), but the product was very weak. There were also some smears in this product, indicating a non-specific product. It will not be used for cloning. ==
 * == PCR #3: Genotyp buffer, primers, and dNTPs as well as Taq polymerase made in Aprell' lab. This reaction did not produce any PCR product. ==
 * 1) = **//Aprell decided to repeat all 3 of these reactions (Troubleshoot Step 1) to see if she would get better results. There could of been pipetting errors by the students.//** //She also did a reaction using Genotype buffer, primers and dNTPs as well as undiluted Genotyp Taq (1 microliter).// =
 * == PCR #1: Genotyp buffer, primers, and dNTPs as well as Platinum Taq from Invitrogen Company. This reaction did not produce a PCR product. ==
 * == PCR #2: Genotyp primers only and GoTo Taq Mix from Promega Company. This specialized taq mix already includes buffers, dNTPs and Taq polymerase. This reaction produced a PCR product at ~1Kb (right size), but again the product was very, very weak. There were also some smears in this product, indicating a non-specific product. ==
 * == PCR #3: Genotyp buffer, primers, and dNTPs as well as Taq polymerase made in Aprell' lab. This reaction did produce a good PCR product. ==
 * == PCR #4: Genotyp buffer, primers, and dNTPs as well as Genotyp Taq polymerase. This reaction did produce a good PCR product. This may be due to not diluting the enzyme, as was done in Troubleshoot Step 1. ==