I am trying to create a genomic library for transposon integration profiling in yeast.
Recently I have been getting a very strange fragmentation pattern. It seems that my fragmentation is at the same time too high and too low.
I am currently shearing for 13 cycles, 3 sets of 3, and one set of 4 for 30 seconds on and 30 seconds off. Others using this protocol for different organisms have gotten great fragmentation with a peak at ~350bp, which is what I am aiming for. I have seen this in the last few samples Ive been using to optimize my procedure. At first I thought that maybe consecutive freeze thaws or DNase contamination may be the problem, but even using samples isolated from the night before and immediately put into the -20 showed this fragmentation, despite two attempts.
A quick rundown of my protocol is using zymolase then the epicenter kit to break open the cells and precipitate most of the protein. From there I treat with RNase A for 1 hr, proteinase k for 2 hours, a phenol chlor extraction, chlor back extraction, and finally an ethanol precipitation. The sample on this BioA had 6 ethanol washes in the ethanol precipitation.
Attached is a BioA of my most recent attempt. the first 8 samples are mine the others belong to someone who was sharing the run with me.
The key is the first 4 samples are from one isolation at 300-600 ng of total DNA increasing by 100 ng per sample. The second 4 samples are the same setup just from a different isolation.
If anyone has any advice on how to improve my fragmentation or an explanation of what could be causing the strange fragmentation pattern I would be very appreciative.
Andrew
Recently I have been getting a very strange fragmentation pattern. It seems that my fragmentation is at the same time too high and too low.
I am currently shearing for 13 cycles, 3 sets of 3, and one set of 4 for 30 seconds on and 30 seconds off. Others using this protocol for different organisms have gotten great fragmentation with a peak at ~350bp, which is what I am aiming for. I have seen this in the last few samples Ive been using to optimize my procedure. At first I thought that maybe consecutive freeze thaws or DNase contamination may be the problem, but even using samples isolated from the night before and immediately put into the -20 showed this fragmentation, despite two attempts.
A quick rundown of my protocol is using zymolase then the epicenter kit to break open the cells and precipitate most of the protein. From there I treat with RNase A for 1 hr, proteinase k for 2 hours, a phenol chlor extraction, chlor back extraction, and finally an ethanol precipitation. The sample on this BioA had 6 ethanol washes in the ethanol precipitation.
Attached is a BioA of my most recent attempt. the first 8 samples are mine the others belong to someone who was sharing the run with me.
The key is the first 4 samples are from one isolation at 300-600 ng of total DNA increasing by 100 ng per sample. The second 4 samples are the same setup just from a different isolation.
If anyone has any advice on how to improve my fragmentation or an explanation of what could be causing the strange fragmentation pattern I would be very appreciative.
Andrew
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