Chromosome Separation Attempt 1 (2/16/10)

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Preparation of Isolation Buffer[edit]

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 example, for 100mM MgSO4:

1g MgSO4 * (1 mol MgSO4/ 120.366g MgSO4) = 

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

Note: We were unable to test the pH of the solutions before adding them to the isolation buffer mix.

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[edit]

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. After this, a syringe with a 22 gauge needle was used to repeat-pipette the samples, hopefully mixing them well.

Results[edit]

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; no individual chromosomes were seen. This shows us that the experiment failed.

File:20X Focus Chrom1.jpg

Image: 20X Magnification

File:40X-2 Focus Chrom1.jpg

Image: 40X Magnification

Observations[edit]

One possible explanation for the apparent failure could be the filtration of the Triton X-100. The liquid is highly viscous, making it extremely hard to pipette and to filter. It's possible that I forced the X-100 through the filter too quickly, cracking the filter. Another possibility, since Sam had similar results with a different procedure, is that the cell cultures didn't grow properly, and so the chromosomes were not fully formed. If the cells didn't grow properly, the colcemid would not have a chance to arrest the cell development in the correct phase, producing DNA that is not in the organized chromosome shapes.