(Under construction) General guidelines Typical PCR conditions for a 20µl reaction (Genomic DNA) Completely thaw, vortex, and briefly spin all reagents EXCEPT Taq polymerase. You can gently mix Taq by inverting or flicking the tube. |Reagent|Volume in µl| |---:|---| |Water|11.2| |10x Buffer| 2| |12x dNTP (2.5mM each)| 1.6| |10x FW Primer (10 µM)|2| |10x RV Primer (10 µM)|2| |100x Homemade Taq Polymerase*1 |0.2| |Template DNA|1| *1 If necessary, add 1/20 vol of Takara Ex Taq in your master mix (do not premix with homemade TAQ) (by Kazu, Aug 22, 2017). - Keep everything on ice or cooling block. - Design PCR (water control is mandatory). - Make master mix (10% more than needed). - Aliquot master mix and add template etc. - Spin down by salad spinner or mini-centrifuge. - Hot start PCR. - To avoid PCR product contamination into lab chemicals in other rooms, use gel room pipets for gel loading. Do not use pipets in other room for gel loading. Conversely do not use the gel room pipets in other rooms. |Step|Temp|Time| |---:|---|---| |1. Initial denaturation| 95˚C | 3 minutes| |2. Denature| 95˚C| 30 seconds | |3. Anneal|55˚C| 30 seconds| |4. Extend| 72˚C| 1 min/kb| |5. Cycle| |Repeat 2-5 35 times| |6. Final extension| 72˚C|5 minutes| |7. Hold|12˚C|Forever| (Plasmid DNA) Troubleshooting contamination - Be aware of aerosolized DNA! Each time you flick a tube of amplified PCR-product open or pipette it, small amounts of DNA become aerosolized and can contaminate future reactions - PCR amplicons can be persistent and we have had problems with them in the past - Try opening your tubes gently, rather than flicking them, and be mindful of handling amplified PCR product outside of the PCR/gel room - Try to handle PCR reagents in a separate space than DNA - Avoid using the same gloves/lab coat for handling post-PCR products and pre-PCR set up - Use filter tips - Try spraying bench with 10% bleach solution, wait 5 minutes, then clean up with water / 70% ethanol (Bleach is corrosive and must be washed away)