Chromosome Separation Attempt 3 (4/5/10)
Notes[edit]
- GM18506 cells used
- Prepared fresh isolation buffer
- Due to lack of RNAse, used 0.9mL isolation buffer instead of full 1mL
- Couldn't pipette the filtered triton X-100
--> Used unfiltered triton X-100
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 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[edit]
6 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. Removed as much supernatant as possible without dislodging the palettes.
After 0.9mL 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 10x
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 distinct masses, though few fully intact chromosomes. 2 of the 6 samples are shown below.
Image: GM18506 Tube 2, 10X Magnification
Image: GM18506 Tube 5, 10X Magnification
Observations[edit]
The same procedure was used as before, taking exceptions with those listed in the Notes section. Interestingly, Tube 2 had a far greater density of chromosomes than any other tube did. This is likely completely random, as Sam did not intend to bias the samples when splitting them into smaller tubes. Using unfiltered Triton X-100 solution did not have a noticeable effect on the results.
Because, so far, all the samples have produced mainly broken-up chromosomes rather than full chromatids, we will try a more gentler pipetting during the next experiment. It is reasonable to conclude that mechanical forces are breaking up the chromosomes, and the main source of any force in this procedure is the pipetting.