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Chromosome Separation Attempt 2 (2/25/10)
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==Notes== *Rather than prepare fresh isolation buffer, we used the buffer prepared for Attempt 1 on 2/16/10. The buffer solution was kept in room temperature in the interim. From the images taken, it doesn't appear that there was any difference caused by the aged isolation buffer. *Also, in an attempt to reduce the foaming, and possible complications caused thereby, of the solution by repeat pipetting via a syringe and needle, we split the samples into two tubes: one was repeat pipetted via syringe, the other homogenized via the PowerGen125. Neither method seems to have created striking differences, pointing to the conclusion that both methods either mix the solutions well, or that neither method is effective. *All images stained using dapi ==Preparation of Isolation Buffer== Solutions of 100mM MgSO4, 55mM KCl, 5.5mM HEPES, and 120mM DTT were needed. For each (minus the DTT), the number of moles per gram of solid was calculated and the resultant molarity from adding 1mL water was then found. Using this as a starting point, the actual amount of water needed to create solutions of the correct molarity was calculated. For MgSO4: **Insert calculation** Similarly, for KCl: Add 0.4g KCL (solid) to 48.764mL water -> 110mM KCl solution For HEPES: Add 0.4g HEPES (solid) to 152.58mL water -> 11mM HEPES solution Because the stock solution of DTT was far more concentrated (1 M) than the needed concentration, a smaller total volume of DTT was added to the isolation buffer, rather than adding the full 0.25mL of diluted DTT. The combined solution of 55mM KCl and 5.5mM HEPES was created by adding 4.5mL 110mM KCl and 4.5mL 11mM HEPES to a separate container, thereby reducing the concentrations in half, but allowing for a total volume of 9mL In the end, the buffer was made as follows: 1mL 100mM MgSO4 9mL 55mM KCl + 5.5mM HEPES 0.03mL 1M DTT 0.7mL 10000U/mL RNase The solution was then filtered via a 10mL syringe with a 22um filter attached. The filtration rate was slow - drop by drop - as to be expected from a filter. ==Isolation of Chromosomes== 2 tubes of cells were extracted and used for this procedure. After being spun down, the palettes formed by the cells were very small and easily disturbed. After removing the majority of the supernatant, the cells tended to re-suspend in the remaining fluid, necessitating another spin in the centrifuge to re-form the palette. After this second spin the remainder of the supernatant could be pipetted out safely. After 1mL of the isolation buffer was added to each tube, the cells were easily resuspended via a few flicks to each tube. While the tubes sat at room temperature, the Triton X-100 filtration was performed. The X-100 filtration: The filter was added to the syringe first, the plunger was removed from the syringe, 500uL Triton X-100 solution was added to the syringe, the X-100 was then filtered (rather forcefully) *Each attempt to push the X-100 solution through the filter caused a large snapping sound, presumably from the filter *500uL was taken from the stock bottle, and only 100uL filtered out of the syringe *The solution is highly viscous, causing troubles when it was pipetted into the syringe and then to each tube The samples were allowed to sit at room temperature for 10 minutes. One sample was mixed via repeat-pipetting, using a syringe with a 22 gauge needle attached. The solution was pipetted 20x The second sample was mixed using the PowerGen125 Homogenizer for ~60 seconds. ==Results== After the samples were incubated in the water bath at 37C for 30min, they were stained and placed under the microscope. The images showed somewhat globular masses. Again the experiment seems to have failed. [[image: 10X_Focus_Chrom2_Repeat.jpg]] Image: Repeat-Pipetted Sample, 10X Magnification [[image: 20X_Focus_Chrom2_Homogenized.jpg]] Image: Homogenized Sample, 20X Magnification [[image: 40X_Focus_Chrom2_Homogenized.jpg]] Image: Homogenized Sample, 40X Magnification ==Observations== Nearly the exact same procedure was followed as in attempt 1, this time allowing for two different mixing techniques. Yet the result was the same: indecipherable masses not at all resembling those of typical chromosomes. It appears that this problem traces back to the cell cultures and the inability of the colcemid to fix the cells in the metaphase state. There are two possible reasons for this: 1) The cells are not growing well and are therefore unable to reach metaphase or 2) the colcemid is failing to work. To fix any problems with cell growth we must re-culture a new cell line and be sure to maintain it properly. If the colcemid is not working, it is either because the incubation time is inaccurate or that the chemical itself is failing to work. Since other groups have been able to get results using colcemid, this leads me to believe that the colcemid does indeed function, but that we are either not incubating the cells in the colcemid for the proper amount of time, or - more likely - the cells are poorly maintained and are therefore not growing properly.
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