Talk:20.109(S10):Aptamer binding assay (Day7): Difference between revisions
Pei-Ann Lin (talk | contribs) (→T/R) |
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|Orange | |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. | ||
|- | |- | ||
|Yellow | |Yellow | ||
Revision as of 12:33, 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% ; 51.4% | 117%; 127% | We had to use extra '6-5' because we ran out of it. Our results came in harmony with expected. We had peak shifts. There was something definitely wrong with our '8-12' pre absorbance, which was abnormally low so we relied heavily on the correcting method to get a reasonable value for it. |
| 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 | ||||
| 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 compared to 16 washes, since we seemed to have lost some 8-12 in those extra 8 washes. The horizontal peak shifts on our graph are consistent with our findings based on the vertical shifts. |
| Pink | ||||
| 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. |
| 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. |
| Yellow | ||||
| 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"
