Talk:20.109(S11):Aptamer binding assay (Day7)

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20.109(S11): Laboratory Fundamentals of Biological Engineering

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Contents

Binding Data Files

Group T/R zip data file W/F zip data file
Red Media:S11_M1D7_TR-Red.zip Media:S11_M1D7_WF-Red.zip
Orange Media:S11_M1D7_TR-Orange.zip Media:S11_M1D7_WF-Orange.zip
Yellow Media:S11_M1D7_TR-Yellow.zip Media:S11_M1D7_WF-Yellow.zip
Green Media:S11_M1D7_TR-Green.zip Media:S11_M1D7_WF-Green.zip
Blue Media:S11_M1D7_TR-Blue.zip Media:S11_M1D7_WF-Blue.zip
Pink Media:S11_M1D7_TR-Pink.zip Media:S11_M1D7_WF-Pink.zip
Purple Media:S11_M1D7_TR-Purple.zip Media:S11_M1D7_WF-Purple.zip


Binding Data Results Summary

T/R

* 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"

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.
Green 0.669; 0.724; 0.713
0.709; 0.786; 0.772
N/A; N/A We used Purple group's mixture, and class' for pre 6-5 and pre 8-12. Since the pre 6-5 and pre 8-12 had approximately the same values, they contained the same aptamers.
Pink 0.62, 0.55, 0.55
0.547, 0.553, 0.56
%2, n/a Washes conducted: 4 and 24 Our graph shows no shift in absorbance peaks and so there is not a significant enough difference in absorbance values to calculate the actual percentages of pre, and post washes. This may be due to experimental error or

also indicate that having an initial percentage of 2% 8-12 is too low to start the SELEX process with and we would ideally want to use more

Binding Curves - Pink
Binding Curves - Pink
Blue

0.0730 0.0150 0.0030 0.0300 0.0430

2% (actual 2.9%) Washes:4(40%) and 32(47%) The shifts in absorbance are shown in the attached graph and are within our expectations. The post wash solutions showed enrichment but more washes correlated to only a slight increase in 8-12 binding.
Binding Curves - Blue
Binding Curves - Blue
Purple 0.36549, 0.455, 0.393628
0.379314, 0.41143137, 0.416490196
10%, n/a Washes conducted: 49.6% (for 8 washes), 57.8% (for 32 washes) The percentages of the post- are higher after the 32 washes than in the 8 washes, suggesting that more washes can be done to obtain greater concentrations of 8-12 collected. The 8-12 pre curve is vertically higher than the 6-5 pre curve, which is consistent with expections. However, the post- curves were expected to be between the the two curves, but they turned out to exceed the 8-12 pre- curve.
Binding Curves - Purple
Binding Curves - Purple
Yellow 0.334, 0.365847682, 0.368622517
0.335192053, 0.351615894, 0.356927152
1%, N/A N/A, N/A The absorbance reading for the pre-mixture was lower than each of the individual 6-5 and 8-12 pre-samples, making it impossible to do a linear extrapolation to calculate the post-wash percentages. We also used 6-5 and 8-12 samples from the blue group and faculty because we did not obtain enough after purification on Day 4. Also, when diluting our samples to 8 micromolar samples before denaturing on Day 7, we accidentally diluted our sample in water instead of selection buffer, and later had to compensate for it by adding equivalents of 2x SB. This reduced the number of moles of our samples in half, and this is reflected in our low absorbance readings.
Red .7065; .707; .7069
.748; .7071; .7045
.5%, N/A N/A, N/A Same as Yellow Group basically. Can't do an extrapolation. We didn't mix ours with water but something obviously happened.
Orange 0.604; 0.607; 0.686; 0.648; 0.662; 0.670 50%, 51.898% 69.620%; 79.746% The calculated amount of 8-12 in the "pre" mixture is 51.898%, which is very close to the theoretical amount (50%) - the difference is probably due to experimental error. Our "post" values for 8-12 were 69.620% for 8 washes, and 79.746% for 16 washes - this shows an increase in 8-12 enrichment compared to the initial 50-50 mix. However, this result is somewhat surprising as the previous group that performed this experiment had a decrease in 8-12 enrichment with increasing number of washes. We had to normalize the curves, since the initial "heme alone" curve was too low and the "post" 8 washes curve was too high. In the normalized graph, the post 16 washes and 8-12 "pre" were very similar (highest); the 6-5 "pre" and "heme alone" were very similar (lowest), and both the mixture "pre" and post 8 washes were in the middle.
Binding Curves - Orange
Binding Curves - Orange


[[Media:Media:Example.ogg]] Media:Example.ogg

W/F

* 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"

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.
Green 0.655; 0.655; 0.737
0.656; 0.668; 0.681
0.25%, 1.21% 16.3%, 32.3%
Binding Curves GREEN
Binding Curves GREEN
Blue 0.753; 0.753; 0.833
0.765; 0.830; 0.816
10%, 15.72% 2 washes: 96.05%; 34 washes: 78.53% We used the T/R Orange group's 6-5 to make our 10% 8-12 mixtures. We used 6-5 (pre) as our base line for correction.
Orange 0.649; 0.638; 0.744
0.632; 0.637; 0.736
2%, <0% ? 0, 92.1% Pre mixture measured lower than 6-5 pre.
Due to experimental error, 2 wash measured lower than 6-5 pre.
Absorbance Curves Orange
Absorbance Curves Orange
Purple 0.717; 0.729; 0.802
0.760; 0.780; 0.769
50%, 42.47% 69.86%, 54.79% On Day 4, we used 6-5 and 8-12 from the orange, green, and faculty as well as some of ours, because after purification we didn't have enough of either one. This could have caused our lower initial concentration.
Binding Curves - Purple
Binding Curves - Purple
Red 0.687; 0.687; 0.770
0.689; 0.734; 0.744
2%, 2.88% 57.31% (4 washes), 69.42% (24 washes) We used 6-5 from orange, green, and faculty because our 6-5 concentration was too low.
Binding Curves RED
Binding Curves RED
Yellow 0.653; 0.654; 0.751
0.709; 0.723; 0.733
50%, 56.27% 70.68%, 81.62% We only used our own RNA.
Binding Curves Yellow
Binding Curves Yellow
Pink 0.591; 0.583; 0.669
0.598; 0.697; 0.644
10%, 17.4% 131.8%, 70.6% The 8 washes result is skewed because of an overall increase in abs at all frequencies. We measured multiple times on the spec. We believe the 16 wash is a more accurate result
Binding Curves Pink
Binding Curves Pink
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