Thursday, June 23, 2011
Week 6- Day 4
Today, we took a step ahead: getting the NCP to run through the gel almost perfectly. Looking at the picture beside, the first column is the DNA ladder and the bold band at the middle stands for 100 base pairs. But we need about 150 base pairs so we can count the bands after the 100bp band to see the 150bp mark because the bands in increments of 10. The next column stand for the NCP digest for 0 minutes, the next for 5minutes, then 10,20 up till 50 minutes. If you notice closely, there is a thick band on the same level of the 150bp on the 5minute digest column. This is an indication that 5 minutes was the adequate time to digest the NCP.We do not use the other columns because there is a lot of smearing of the whitish band and this indicates over digesting. Therefore we digested our NCP for only 5 minutes, and with the packing ready, we are currently running our NCP through it to separate the mono-nucleosomes from the di-nucleosomes using the size-exclusion chromatograhy technique. This way, the mono-nucleosomes we actually need run faster than the di-nucleosomes through the packing and we can collect them. The NCP has not finished running through but I will keep an eye on it till it does.