Talk:20.109(S10):Aptamer binding assay (Day7): Difference between revisions
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|4 washes 53.5%;24 washes 92.4% | |4 washes 53.5%;24 washes 92.4% | ||
|We hypothesized that with 4 washes the final mixture would be 60% 8-12, and that for 24 washes the final mixture would be 90% 8-12. Our observed values are relatively close. We were suprised that the pre-mixtue had only 10% 8-12 when we though we put in 50% 8-12. We are trying to figure this out. | |We hypothesized that with 4 washes the final mixture would be 60% 8-12, and that for 24 washes the final mixture would be 90% 8-12. Our observed values are relatively close. We were suprised that the pre-mixtue had only 10% 8-12 when we though we put in 50% 8-12. We are trying to figure this out. | ||
Raw Data:[[Media:raw. | Raw Data:[[Media:raw.pdf]] | ||
Normalized Data:[[Media:normalized.pdf]] | Normalized Data:[[Media:normalized.pdf]] | ||
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Revision as of 15:32, 6 March 2010
Binding Data Files
| Group | T/R zip data file | W/F zip data file |
|---|---|---|
| Red | Media:S10_M1D7_TR-Red.zip | Media:S10_M1D7_WF-Red.zip |
| Orange | Media:S10_M1D7_TR-Orange.zip | Media:S10_M1D7_WF-Orange.zip |
| Yellow | Media:S10_M1D7_TR-Yellow.zip | Media:S10_M1D7_WF-Yellow.zip |
| Green | Media:S10_M1D7_TR-Green.zip | Media:S10_M1D7_WF-Green.zip |
| Blue | Media:S10_M1D7_TR-Blue.zip | Media:S10_M1D7_WF-Blue.zip |
| Pink | Media:S10_M1D7_TR-Pink.zip | Media:S10_M1D7_WF-Pink.zip |
| Purple | Media:S10_M1D7_TR-Purple.zip | Media:S10_M1D7_WF-Purple.zip |
Binding Data Results Summary
T/R
| Group | Corrected A405 values* | Pre % 8-12: expected, measured | Post % 8-12: fewer washes, more washes (measured) | Additional comments |
|---|---|---|---|---|
| Syntax sample | 0.1; 0.2; 0.3 0.4; 0.5; 0.6 |
10%, 12% | 40%, 50% | We used Grey group's 6-5 because we didn't have any. |
| red | 0.278; 0.281; 0.0.298; 0.299; 0.35, 0.377 |
50% ; 50.7% | 111%; 116% | We had to use extra '6-5' because we ran out of it. Our results came in harmony with what expected in terms of peak shifts. There was something definitely wrong with our '8-12' and 'pre-wash' mixture absorbance, which were abnormally low (even lower than '6-5' peak we tried to fix it by making the 350 nm - 385 nm overlap by finding the average distance, but that procedure neither made them overlap nor did it increase the absorbance peak. 
|
| orange | 0.181392157 0.182 0.236803922 0.18354902 0.256019608 0.281509804 |
2%; 3.3696% | 4 washes: 134.8675%; 24 washes: 181.1195% |
Only used .1nmols each for the binding assay samples
Normalized Data: Media:NormalizedAbsorbance.png |
| Yellow | 0.270, 0.276, 0.345, 0.272, 0.325, 0.329 | 2%; -5.79% | 8 washes: 71.01%, 16 washes: 76.80% | The pre value is definitely wrong. I believe it is due to either the corrections or OpenOffice dropping sig-figs somewhere. Our post mixtures are around what I would expect. The horizontal peak shifts seem to be consistent with our findings. File:Results chart.pdf |
| Green | 0.216, 0.217, 0.292, 0.227, 0.258, 0.283 | 10%; 13.333% | 4 washes: 65.667%; 24 washes: 88.000% |
File:20.109 assay graphs.pdf Due to the corrections, the heme and 6-5 aptamer horizontal peaks are a little off; the 6-5 aptamer graph should be shifted up from the heme graph at low absorbances, but instead, it's simply lined up with the heme graph. The other graphs seem consistent with the fact that 8-12 graph should be higher horizontally than the 6-5 graph, with the heights increasing in the following order: mix 10% pre, mix 10% post 4x, post 24x, and 8-12 aptamer. |
| Blue | 0.261, 0.263, 0.322, 0.294, 0.3111, 0.306 |
50%; 52% | 8 washes: 80%; 16 washes: 72% |
The difference in observed % 8-12 in our pre-selection mix versus our expected value of 50% could be due to pipetting errors, and the fact that we normalized the absorbance values to our 6-5 pre-selection sample. The values for our post-selection 8-12 percentages are along the lines of what we expected. It seems like 8 washes is closer to the ideal number of washes. The horizontal peak shifts on our graph are consistent with our findings based on the vertical shifts. |
| Pink | 0.249; 0.248; 0.317; 0.257; 0.321; 0.294; | 10%, 14% | 8 washes: 67% 16 washes: >100% |
Our data has two major errors. 1) Our graph shows that we have over 100% 8-12 in our 16 wash sample. We believe that this is due to an error in the molecular weight assumption that we made. We believe that we overestimated the MW which caused us to not dilute the sample enough. This means we had over 8 M sample which we then mixed with 6M samples, assuming we probably had less than actually 8M. This caused our absorbance to be very high. 2) We added double heme to our 8 wash mixture. We demed that the absorbance we found from that sample was useless, and we ran another sample with our remaining aptamer 8 wash mixture. It contained 100ul mixture 75 ul buffer and 175 ul heme. We found the expected ratio of heme to mixture in nanomoles and then corrected our ratio and data accordingly. This is the data shown on the graph.
File:NormalizedGraphjpg Our percent 8-12 aptamer in the mixture was 14% instead of 10%, this is pretty close and the error could be due to pipetting. Our post values of 8-12 for the 8 washes was relatively normal but our 16 wash was too high, as we explained above. Our peaks were consistent, with the exception of our 8 wash corrected data which did not shift as expected. |
| Purple | 0.254; 0.259; 0.3559 0.2971; N/A; N/A |
50%, 41.5% | N/A | We did not obtain proper data for the mixes post-wash, unfortunately. Also, our 8-12 "pre" sample came from the Blue group. <3 The horizontal and vertical shifts for the samples we did have all appear consistent with our expected results. Corrected Data: Media:Yuan-mod1spec-CORRECTED.png |
* Be sure to post the values you got after baseline correction, not the raw data (if your curves didn't line up)!
* Order: heme, 6-5, 8-12, "pre" mix, post low wash, post high wash
* Recommended syntax for A405 column: "#1 ; #2; #3 <br> #4; #5; #6"
W/F
| Group | Corrected A405 values* | Pre % 8-12: expected, measured | Post % 8-12: fewer washes, more washes (measured) | Additional comments |
|---|---|---|---|---|
| Syntax sample | 0.1; 0.2; 0.3 0.4; 0.5; 0.6 |
10%, 12% | 40%, 50% | We used Grey group's 6-5 because we didn't have any. |
| Red | 0.260;0.252;0.323;0.262;0.311;0.30888 | 10%, 3.5% | 8 Washes 81%, 16 Washes 78% | Trying to figure out what went wrong with premix ratios.
Raw Data: Media:WFRed-UV.png |
| Orange | 0.2802;0.28;0.362;0.289;0.324;0.3558 | 50%;10% | 4 washes 53.5%;24 washes 92.4% | We hypothesized that with 4 washes the final mixture would be 60% 8-12, and that for 24 washes the final mixture would be 90% 8-12. Our observed values are relatively close. We were suprised that the pre-mixtue had only 10% 8-12 when we though we put in 50% 8-12. We are trying to figure this out.
Raw Data:Media:raw.pdf Normalized Data:Media:normalized.pdf |
| Yellow | 0.2623; 0.2580; 0.3333 0.2620; 0.2895; 0.2863 |
2%;5.4% | 8 Washes 41.8%; 16 washes 37.6% | During the dilution step, we accidentally diluted it to 7.7uM as opposed to the 8uM (This is estimating final concentration to be 50% 8-12) However, we corrected this by decreasing the amount of heme so that it was still a 1:1 ratio. Therefore, the results should not have been affected. Normalization was used to correct for the heme curve, which was lower than all the other curves. Enrichment values were close to expected ones (50%) but slightly surprising that 8 washes enriched a bit more than 16 washes.
Raw Data: Raw Data: Media:Absorbance data.jpg |
| Green | 0.248; 0.245; 0.313; 0.253; 0.289; 0.287 |
10%, 12% | 64%, 61% | Before normalization of the Absorbance curves, the pre8-12 was much higher than the pre6-5 in the 380-385 region.
Our pre mix % was close to expected (~11.9%). Our hypothesized post values were different from the actual ones. We were surprised to see that the 8x sample had enrichment and that 16x showed decrease in enrichment. We hypothesized that the 8x sample would have 50% 8-12 and the 16x sample would have 80% 8-12. Relative positions of peaks are as expected. (Greater vertical shift corresponds to greater horizontal shift). Raw Data: Media:RawBindingAssayM1D7.png |
| Blue | ||||
| Pink | ||||
| Purple |
* Be sure to post the values you got after baseline correction, not the raw data (if your curves didn't line up)!
* Order: heme, 6-5, 8-12, "pre" mix, post low wash, post high wash
* Recommended syntax for A405 column: "#1 ; #2; #3 <br> #4; #5; #6"
