Chromosome Separation Trials: Difference between revisions
>Jbrubake (Created page with '==Preparation of Isolation Buffer== '''The same concentration buffer was used for each experiment:''' Solutions of 100mM MgSO4, 55mM KCl, 5.5mM HEPES, and 120mM DTT were needed…') |
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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: | 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) = | 1g MgSO4 * (1 mol MgSO4/ 120.366g MgSO4) = 8.308M | ||
Want 100mM, so take 0.20 grams MgSO4 and add to 16.62mL water | |||
Similarly, for KCl: | Similarly, for KCl: |
Latest revision as of 00:46, 29 May 2010
Preparation of Isolation Buffer[edit]
The same concentration buffer was used for each experiment:
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) = 8.308M
Want 100mM, so take 0.20 grams MgSO4 and add to 16.62mL water
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.
2/16/10[edit]
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.
Image: 20X Magnification
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.
2/25/10[edit]
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.
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.
Notes[edit]
- 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 2x Dapi
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. Again the experiment seems to have failed.
File:10X Focus Chrom2 Repeat.jpg
Image: Repeat-Pipetted Sample, 10X Magnification
File:20X Focus Chrom2 Homogenized.jpg
Image: Homogenized Sample, 20X Magnification
File:40X Focus Chrom2 Homogenized.jpg
Image: Homogenized Sample, 40X Magnification
Observations[edit]
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.
4/5/10[edit]
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
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
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.