All right, it is likely that you may need to repeat the same PCR reaction about oh I don’t know how many times before you can get enough of what you want. I have been told, but was resilient to admit that contamination can and WILL occur if you don’t take precautions.
- Do not talk while you do your PCR.
- Do not wave stuff around your PCR tube or samples or Primers or any of the solutions you’re working on because a spec of dust can cause a strange band in your result after you go and analyze your reaction.
- Do not stoop over or lean over to look into your PCR tubes, instead tilt the tubes toward you so you can pipet into it.
- Mix every solution really well and make sure to centrifuge it down so that there is no residual solution on the side of your tubes or in your cap. I recently lost a lot of DNA because of residual solution in my cap.
- Make sure everything that you use is autoclaved and as clean as can be.
- Consider running a gradient PCR, which means the same reaction mix, but at varying temperature (+/- 0.2 to 1.0 C) to assess the best annealing temperature for your primers
- Clean up your PCR by doing a Gel PCR purification or use a kit such as Qiagen MinElute PCR Purification, or Qiaquick PCR Purification (depending on the size of your sequence and of what base pairs you are trying to remove) to clean up your PCR. Keep in mind that gel purification is likely to lower your actual concentration of amplified sequence. In other words, if you have a wee bit to begin with, it might not be worth it to do the clean up.
All in all, be very very mindful of what you are doing. Read your protocols way before you do your experiment and have the necessary calculations ready BEFORE you do your experiment.
Good luck and happy experimenting!