IGEM:Harvard/2006/Adaptamers/Notebook/2006-8-26

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(Strand Displacement)
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1. Standard setup: 40 pmol A20 + 100uL .4% solids SMB + 160 pmol thrombin
1. Standard setup: 40 pmol A20 + 100uL .4% solids SMB + 160 pmol thrombin
 +
2. =1
2. =1
 +
3. +10 pmol A20.20
3. +10 pmol A20.20
 +
4. + 20 pmol A20.20
4. + 20 pmol A20.20
 +
5. + 40 pmol A20.20
5. + 40 pmol A20.20
 +
6. + 10 pmol A20.35
6. + 10 pmol A20.35
 +
7. + 20 pmol A20.35
7. + 20 pmol A20.35
 +
8. + 40 pmol A20.35
8. + 40 pmol A20.35
 +
9. r A20 A50
9. r A20 A50
 +
10. r A20 T20
10. r A20 T20
 +
11. r A20 S20
11. r A20 S20
 +
12. r thrombin nothing
12. r thrombin nothing
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*Results
*Results
 +
A2. Standard setup: 40 pmol A20 + 100uL .4% solids SMB + 160 pmol thrombin. Intensity = 15776.12.
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A4. =1. Intensity = 1476.3.
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A6. r A20 A50. Intensity = 952.49.
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 +
A8. +10 pmol A20.20. Intensity = 1215.66.
 +
 +
A10. + 20 pmol A20.20. Intensity = 2292.83.
 +
 +
A12. + 40 pmol A20.20. Intensity = 3611.68.
 +
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C2. + 10 pmol A20.35. Intensity = 2419.15.
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C4. + 20 pmol A20.35. Intensity = 5133.35.
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C6. + 40 pmol A20.35. Intensity = 988.82.
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C8. r A20 T20. Intensity = 1483.64.
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D10. r A20 S20. Intensity = 10617.96.
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C12. r thrombin nothing. Intensity = 1889.81.
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 +
 +
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[Image:Cst8-26replacement.jpg]]
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 +
These results were completely loopy. The positive controls in A4 had a very low intensity; the negative control in D10 had a very high intensity. It is possible that samples were mixed up. Addition of displacement strands resulted in relatively low fluorescence; however addition of more displacement strand tended to increase intensity (A8-A12, C2-C6). All in all puzzling data; the experiment must be run again.
=== ELISA ===
=== ELISA ===
We'll attempt to quantify how much thrombin is bound by the adaptamers using an ELISA
We'll attempt to quantify how much thrombin is bound by the adaptamers using an ELISA

Revision as of 21:01, 29 October 2006

8/26

Strand Displacement

We'll test if strand displacement can quench the adaptamers.

  • Abbreviations/Syntax

SMB: Streptavidin Magnetic Beads BB: Bittker Buffer SS: standard setup r A B: replace A in SS with B.

  • Conditions

1. Standard setup: 40 pmol A20 + 100uL .4% solids SMB + 160 pmol thrombin

2. =1

3. +10 pmol A20.20

4. + 20 pmol A20.20

5. + 40 pmol A20.20

6. + 10 pmol A20.35

7. + 20 pmol A20.35

8. + 40 pmol A20.35

9. r A20 A50

10. r A20 T20

11. r A20 S20

12. r thrombin nothing

  • Methods

100 uL SMB --> 10 uL + 40pmol (1-8, 12. A20 9. A50 10. T20 11. S20 12. A20) + (1-11. 160 pmol thrombin) --> 15 uL -->30 minute shake --> + (3. 10 pmol A20.20 4. 20 pmol A20.20 5. 40 pmol A20.20 6. 10 pmol A20.35 7. 20 pmol A20.35 8. 40 pmol A20.35) --> 19 uL --> shake 20 minutes --> 2X wash --> + 4 uL .200 ug/uL anti-thrombin primary Ab --> 2X wash --> + 1 ug/uL seconday Ab --> 3X wash --> 50 uL --> imaging on 96 well plate.

  • Results

A2. Standard setup: 40 pmol A20 + 100uL .4% solids SMB + 160 pmol thrombin. Intensity = 15776.12.

A4. =1. Intensity = 1476.3.

A6. r A20 A50. Intensity = 952.49.

A8. +10 pmol A20.20. Intensity = 1215.66.

A10. + 20 pmol A20.20. Intensity = 2292.83.

A12. + 40 pmol A20.20. Intensity = 3611.68.

C2. + 10 pmol A20.35. Intensity = 2419.15.

C4. + 20 pmol A20.35. Intensity = 5133.35.

C6. + 40 pmol A20.35. Intensity = 988.82.

C8. r A20 T20. Intensity = 1483.64.

D10. r A20 S20. Intensity = 10617.96.

C12. r thrombin nothing. Intensity = 1889.81.


[Image:Cst8-26replacement.jpg]]

These results were completely loopy. The positive controls in A4 had a very low intensity; the negative control in D10 had a very high intensity. It is possible that samples were mixed up. Addition of displacement strands resulted in relatively low fluorescence; however addition of more displacement strand tended to increase intensity (A8-A12, C2-C6). All in all puzzling data; the experiment must be run again.

ELISA

We'll attempt to quantify how much thrombin is bound by the adaptamers using an ELISA

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