User talk:Brian P. Josey/Notebook/2009/12/04
Steve Koch 23:22, 4 December 2009 (EST): I am not super-familiar with how the SYBR-safe gels look yet. But these look very smeary to me. I wonder if it's because the 5 minute extension is a bit long for 1.1 kb products? Or it could, perhaps, just be sub-optimal magnesium (or other conditions). If we were doing quantitative things with the dig / bio products, it would be a little worrying, because a smear could indicate a spread of product lengths, which would them mean our tethers would not have the contour length we expect. In your case, these could still be useful, since I'm guessing you're using them just as a means of diagnosing whether tethering is working. In any case, great work and great notebook these past few days!
Brian P. Josey 19:20, 6 December 2009 (EST) I was concerned about the smearing too. To be completely honest, I am not a hundred percent there yet on reading gels, so thanks for the input. Do we usually have a set time for running the gel based on the length of the DNA, or is it something that you just go with whatever is convenient? I think that we will still be able to use what we have, but I'll check with Ant. If he wants to, we could optimize the reaction some more and find a way to get rid of the smearing.
- Steve Koch 20:43, 7 December 2009 (EST):Actually, I didn't mean the gel was run to long, but rather that perhaps the elongation step (72C) of the PCR reaction was too short for the short products. Also, FYI: The long reaction (4.4 kb) tends to be smeary without the "perfect match" expensive product that we used to use. But the short reaction (1.1 kb) tended to work well without smearing.