Biomod/2011/Caltech/DeoxyriboNucleicAwesome/Gel Experiments

Thursday, April 18, 2024 Home Members Project Protocols Progress Discussion References

Design for the Gel Experiments See also: List of Strands Example Experiment 0-1 The number to the left of the dash in the title represents which block of tests it belongs to, and the number to the right of the dash represents it's identity within the block Gel Identity: The number to the left of the dash in this entry represents which gel the experiment will be observed on, and the right of the dash represents which lane it will occupy. Equation: This shows the reagents and expected outcomes. Purpose: This explains why this particular experiment is necessary or what it shows. Controls: These are the controls necessary to read the results. Positive Result: This is what we should observe to verify that this particular reaction worked as intended. Negative Result: This is what we expect not to see. If this occurs, we will need to explore further for the cause of failure. List of Experiments X + Y indicates strand X and strand Y exist independently in the same solution. (X - Y) indicates that strand X and strand Y bind to form a complex. Block 1: Walker Binding to track Experiment Design 1-1 Gel Identity: 1-6 Equation: W + TR1 → (W - TR1) Purpose: To show that the Walker binds to one of the track strands Controls: W, TR1 Positive Result: We should see a dark band not corresponding to either of the controls. We might see faint bands corresponding with the controls. This indicates that the two strands did in fact bind. Negative Result: We should not see two dark bands corresponding to the control lanes. This indicates that the strands did not bind. Experiment Design 1-2 Gel Identity: 1-7 Equation: W + TR2 → (W - TR2) Purpose: To show that the Walker binds to the other track strand Controls: W, TR2 Positive Result: We should see a dark band not corresponding to either of the controls. We might see faint bands corresponding with the controls. This indicates that the two strands did in fact bind. Negative Result: We should not see two dark bands corresponding to the control lanes. This indicates that the strands did not bind. Experiment Design 1-3 Gel Identity: 1-12 Equation: TR1 + TR2 → TR1 + TR2 Purpose: To show that the Tracks do not bind to each other. Controls: TR1, TR2 Positive Result: Because TR1 and TR2 are the same length, we should only see one dark band at the same position as those in the control lane. Negative Result: We should not see one dark band positioned differently than the bands in the control lanes. This indicates that the strands bound in a way that we did not intend. Block 2: Walker Triggering Mechanism Experiment Design 2-1 Gel Identity: 1-8 Equation: W + WI → (W - WI) Purpose: To show that the Walker Inhibitor Binds to the Walker Controls: W, WI Positive Result: We should see a dark band not corresponding to either of the controls. We might see faint bands corresponding with the controls. This indicates that the two strands did in fact bind. Negative Result: We should not see two dark bands corresponding to the control lanes. This indicates that the strands did not bind. Experiment Design 2-2 Gel Identity: 1-9 Equation: WI + WT → (WI - WT) Purpose: To show that the Walker Inhibitor Binds to the Walker Trigger Controls: WI, WT Positive Result: We should see a dark band not corresponding to either of the controls. We might see faint bands corresponding with the controls. This indicates that the two strands did in fact bind. Negative Result: We should not see two dark bands corresponding to the control lanes. This indicates that the strands did not bind. Experiment Design 2-3 Gel Identity: 1-10 Equation: W + WI + TR1 → (W - WI - TR1) Purpose: To show that the Track binding to the Walker does not interfere with the binding between the Walker and Walker Inhibitor Controls: W, WI, TR, (W - WI), (W - TR1) Positive Result: We should see a dark band not corresponding to any of the controls. We might see faint bands corresponding with the controls. This indicates that the two strands did in fact bind. Negative Result: We should not see dark bands corresponding to any of the control lanes. This indicates that the strands did not bind as intended. The controls should give us enough information to determine which (if any) of the bindings is preferred by the system. Experiment Design 2-4 Gel Identity: 1-11 Equation: (W - WI - TR1) + WT → (W - TR1) + (WI - WT) Purpose: To show that the Walker Trigger strips the Walker inhibitor off of the Walker Controls: W, WI, WT, TR1, (W - WI), (W - TR1), (W - WI - TR1) Positive Result: We should see dark bands corresponding to two of the control lanes of the products of this reaction( (WI - WT) and (W - TR1) ). We may see light bands corresponding to the reagents. Negative Result: We should not see dark bands corresponding to any other control lanes. This indicates that the strands did not bind as intended. The controls should give us enough information to determine which (if any) of the other bindings is preferred by the system. Block 3: Random Walking Mechanism Experiment Design 3-1 Gel Identity: 2-5 Equation: TR2 + PTR2 → (TR2 - PTR2) Purpose: To show that Track 2 binds with its probe. This will allow us to distinguish between Track 2 and Track 1 in future experiments. Controls: TR2, PTR2 Positive Result: We should see a dark band different from either of the controls. We may see light bands corresponding to the reagents. Negative Result: We should not see dark bands corresponding to either of the controls. This indicates that the strands did not bind. Experiment Design 3-2 Gel Identity: 2-6 Equation: W + (TR2 - PTR2) → (TR2 - PTR2) Purpose: To show that Track 2's probe does not interfere with the binding of the Walker to track 2. Controls: W, TR2, PTR2, (TR2 - PTR2) Positive Result: We should see a dark band different from the controls. We may see light bands corresponding to the reagents. Negative Result: We should not see dark bands corresponding to any of the control lanes. This indicates that the strands did not bind as intended. The controls should give us enough information to determine which (if any) of the bindings is preferred by the system. Experiment Design 3-3 Gel Identity: 2-8 Equation: (W - TR1) + (TR2 - PTR2) ↔ (W - TR2 - PTR2) + TR1 Purpose: To show that the Walker can move between tracks, specifically from Track 1 to Track 2. The system should equalize with walkers on both tracks. Controls: W, TR1, TR2, PTR2, (W - TR1), (TR2 - PTR2), (W - TR2 - PTR2) Positive Result: We should see a dark bands corresponding to all of the reagents and products. Negative Result: We should not see any other dark bands. These would indicate that This indicates that either the Walker did not move as intended or that the movement interfered with another binding. The controls should give us enough information to determine which (if any) of the bindings is preferred by the system. Experiment Design 3-4 Gel Identity: 2-9 Equation: TR1 + (W - TR2 - PTR2) ↔ (TR2 - PTR2) + (W - TR1) Purpose: To show that the Walker can move between tracks, specifically from Track 2 to Track 1. The system should equalize with walkers on both tracks. Controls: W, TR1, TR2, PTR2, (W - TR1), (TR2 - PTR2), (W - TR2 - PTR2) Positive Result: We should see a dark bands corresponding to all of the reagents and products. Negative Result: We should not see any other dark bands. These would indicate that either the Walker did not move as intended or that the movement interfered with another binding. The controls should give us enough information to determine which (if any) of the bindings is preferred by the system. Experiment Design 3-5 Gel Identity: 2-12 Equation: (W - WI - TR1) + (TR2 - PTR2) → (W - WI - TR1) + (TR2 - PTR2) Purpose: To show that the Walker Inhibitor does in fact inhibit walking. Controls: W, TR1, TR2, PTR2, (W - TR1), (TR2 - PTR2), (W - TR2 - PTR2) Positive Result: We should see a dark bands corresponding to only the reagents, since we are looking for no change in the system. Negative Result: We should not see any other dark bands. These would indicate that either the Walker Inhibitor did not work as intended or that the Walker Inhibitor interfered with another binding. The controls should give us enough information to determine which (if any) of the bindings is preferred by the system. Experiment Design 3-6 Gel Identity: 2-13 Equation: (W - WI - TR1) + (TR2 - PTR2) + WT → (WI - WT) + (TR2 - PTR2) + (W - TR1) + (W - TR2 - PTR2) + TR1 Purpose: To show that the Walker Trigger initiates walking. Controls: W, TR1, TR2, PTR2, (W - TR1), (TR2 - PTR2), (W - TR2 - PTR2) Positive Result: We should see a dark bands corresponding to only the products, since these correspond to the result of random walking Negative Result: We should not see any other dark bands. These would indicate that either the Walker Trigger affected the system in some unintended way. The controls should give us enough information to determine which (if any) of the bindings is preferred by the system. Block 4: Cargo Pickup Mechanism Experiment Design 4-1 Gel Identity: 4-4 Equation: C1 + CA → (C1 - CA) Purpose: To show that the Cargo binds to the Cargo Attaching point. Controls: C1, CA Positive Result: We should see one dark band that doesn't correspond to either of the controls. We may see light bands corresponding with the reagents. Negative Result: We should not see two dark bands corresponding to the controls. This indicates that the strands did not bind. Experiment Design 4-2 Gel Identity: 4-5 Equation: W + C1 → (W - C1) Purpose: To show that the Cargo binds to the Walker. Controls: W, C1 Positive Result: We should see one dark band that doesn't correspond to either of the controls. We may see light bands corresponding with the reagents. Negative Result: We should not see two dark bands corresponding to the controls. This indicates that the strands did not bind. Experiment Design 4-3 Gel Identity: 4-6 Equation: W + (C1 - CA) → (W - C1) + CA Purpose: To show that the Walker strips the Cargo off of the Cargo Attaching point. Controls: W, C1, CA, (W - C1), (C1 - CA) Positive Result: We should see two dark bands that correspond to products of the reaction. We may see light bands corresponding with the reagents. Negative Result: We should not see any other dark bands. This indicates that the Cargo did not transfer to the Walker as intended. Experiment Design 4-4 Gel Identity: 4-8 Equation: W + C1 + TR1 → (W - C1 - TR1) Purpose: To show that Track 1 does not interfere with the binding between the Walker and Cargo. Controls: W, C1, TR1, (W - C1), (W - TR1) Positive Result: We should see one dark band that doesn't correspond to any of the controls. We may see light bands corresponding with the reagents. Negative Result: We should not see any dark bands corresponding to the controls. This indicates that Track 1 interferes with the binding between the Walker and Cargo. Experiment Design 4-5 Gel Identity: 4-9 Equation: W + C1 + TR2 → (W - C1 - TR2) Purpose: To show that Track 2 does not interfere with the binding between the Walker and Cargo. Controls: W, C1, TR2, (W - C1), (W - TR2) Positive Result: We should see one dark band that doesn't correspond to any of the controls. We may see light bands corresponding with the reagents. Negative Result: We should not see any dark bands corresponding to the controls. This indicates that Track 1 interferes with the binding between the Walker and Cargo. Experiment Design 4-6 Gel Identity: 4-10 Equation: (W - TR1) + (C1 - CA) → (W - C1 - TR1) + CA Purpose: To show that Track 1 does not interfere with the Walker stripping the Cargo off of the Cargo Attaching point. Controls: W, C1, CA, TR1, (W - C1), (W - TR1), (C1 - CA) Positive Result: We should see one dark band that doesn't correspond to one of the controls(for (W - C1 - TR1) ), and one dark band that corresponds with the remaining strand (CA). We may see light bands corresponding with the reagents. Negative Result: We should not see any other dark bands. This indicates that Track 1 interferes with the binding between the Walker and Cargo. Experiment Design 4-7 Gel Identity: 4-11 Equation: (W - TR2) + (C1 - CA) → (W - C1 - TR2) + CA Purpose: To show that Track 2 does not interfere with the Walker stripping the Cargo off of the Cargo Attaching point. Controls: W, C1, CA, TR2, (W - C1), (W - TR2), (C1 - CA) Positive Result: We should see one dark band that doesn't correspond to one any the controls(for (W - C1 - TR2) ), and one dark band that corresponds with the remaining strand (CA). We may see light bands corresponding with the reagents. Negative Result: We should not see any other dark bands. This indicates that Track 1 interferes with the binding between the Walker and Cargo. Block 5: Goal Triggering Mechanism Experiment Design 5-1 Gel Identity: 3-4 Equation: CG1 + CGI → (CG1 - CGI) Purpose: To show that the apropreate inhibitor binds to the goal. Controls: CG1, CGI Positive Result: We should see one dark band that doesn't correspond to any of the controls. We may see light bands corresponding with the reagents. Negative Result: We should not see any other dark bands. This indicates that the inhibitor does not bind with the goal. Experiment Design 5-2 Gel Identity: 3-5 Equation: CGI + CGT → (CGI - CGT) Purpose: To show that the trigger binds with the inhibitor. Controls: CGI, CGT Positive Result: We should see one dark band that doesn't correspond to any of the controls. We may see light bands corresponding with the reagents. Negative Result: We should not see any other dark bands. This indicates that the inhibitor does not bind with the goal. Experiment Design 5-3 Gel Identity: 3-6 Equation: (CG1 - CGI) + CGT → CG1 + (CGI - CGT) Purpose: To show that the trigger is capable of stripping the inhibitor off of the goal. Controls: CG1, CGI, CGT, (CG1 - CGI), (CGI - CGT) Positive Result: We should see one dark band that corresponds to one of the controls(for (CGI - CGT) ), and one dark band that corresponds with the remaining strand (CG1). We may see light bands corresponding with the reagents. Negative Result: We should not see any other dark bands. This indicates that the trigger does not interract with the inhibitor properly. Block 6: Cargo Drop Off Mechanism Experiment Design 6-1 Gel Identity: 6-7 Equation: C1 + CG1 → (C1 - CG1) Purpose: To show that the cargo binds to the goal. Controls: C1, CG1 Positive Result: We should see one dark band that doesn't correspond to any of the controls. We may see light bands corresponding with the reagents. Negative Result: We should not see any other dark bands. This indicates that the cargo does not bind with the goal. Block 7: Time-based Tests Experiment Design 7-1 Gel Identity: 3-6 Equation: (W - C1) + (CG1 - CGI) → (C1 - CG1) + W + CGI Purpose: There is a potential for 4 way strand displacement to occur with an undesired affect. We wish to find the rate at which this reaction occurs in these timed reactions. Controls: W, C1, CG1, CGI, (W - C1), (CG1 - CGI), (C1 - CG1) Positive Result: Because we are hoping that this reaction doesn't occur, a result that indicates that everything is working correctly would show two dark bands corresponding to (W - C1) and (GC1 - GCI). Negative Result: Any other dark bands would be indicative of the Goal Inhibitor not actually inhibiting the function of the Goal. Block 8: Walker Goal Functionality Experiment Design 8-1 Gel Identity: 8-6 Equation: W + WG → (W - WG) Purpose: To show that the Walker binds to a Walker goal intended to be used in timing the speed of our random walking mechanism. Controls: W, WG Positive Result: We should see one dark band that doesn't correspond to anu of the controls. We may see light bands corresponding with the reagents. Negative Result: We should not see any other dark bands. This indicates that the Walker does not bind to the goal. Experiment Design 8-2 Gel Identity: 8-7 Equation: WG + PWG → (WG - PWG) Purpose: To show that the Walker Goal binds to its probe. This will help us easily distinguish between the walker goal and Track strands. Controls: WG, PWG Positive Result: We should see one dark band that doesn't correspond to any of the controls. We may see light bands corresponding with the reagents. Negative Result: We should not see any other dark bands. This indicates that the Walker does not bind to the goal. Experiment Design 8-3 Gel Identity: 8-6 Equation: W + (WG - PWG) → (W - WG - PWG) Purpose: To show that the Walker Goal Probe does not interfere with the bond between the Walker and Walker goal Controls: W, WG, PWG, (WG - PWG), (W - WG) Positive Result: We should see one dark band that doesn't correspond to any of the controls. We may see light bands corresponding with the reagents. Negative Result: We should not see any other dark bands. This indicates that the Walker Goal Probe interferes with the Walker and Walker Goal bond. Experiment Design 8-4 Gel Identity: 8-10 Equation: (W - TR1) + (WG - PWG) → TR1 + (W - WG - PWG) Purpose: To show that the Walker Goal can strip the Walker off of Track 1. Controls: W, WG, PWG, TR1, (W - TR1), (WG - PWG), (W - WG - PWG) Positive Result: We should see two dark bands that correspond to the controls for TR1 and (W - WG - PWG). We may see light bands corresponding with the reagents. Negative Result: We should not see any other dark bands. This indicates that the Walker Goal is incapable of stripping the walker off of Track 1. Experiment Design 8-5 Gel Identity: 8-11 Equation: (W - TR2) + (WG - PWG) → TR2 + (W - WG - PWG) Purpose: To show that the Walker Goal can strip the Walker off of Track 2. Controls: W, WG, PWG, TR2, (W - TR2), (WG - PWG), (W - WG - PWG) Positive Result: We should see two dark bands that correspond to the controls for TR2 and (W - WG - PWG). We may see light bands corresponding with the reagents. Negative Result: We should not see any other dark bands. This indicates that the Walker Goal is incapable of stripping the walker off of Track 2. Experiment Design 8-6 Gel Identity: 8-12 Equation: TR1 + (W - WG - PWG) → TR1 + (W - WG - PWG) Purpose: To show that the Walker cannot move to Track 1 from the Walker Goal. Controls: W, WG, PWG, TR1, (W - TR1), (WG - PWG), (W - WG - PWG) Positive Result: We should see two dark bands that correspond to the controls for TR1 and (W - WG - PWG). We may see light bands corresponding with the reagents. Negative Result: We should not see any other dark bands. This indicates that the Walker is capable of leaving the walker goal. Experiment Design 8-7 Gel Identity: 8-13 Equation: (W - TR2) + (WG - PWG) → TR2 + (W - WG - PWG) Purpose: To show that the Walker cannot move to Track 1 from the Walker Goal. Controls: W, WG, PWG, TR1, (W - TR1), (WG - PWG), (W - WG - PWG) Positive Result: We should see two dark bands that correspond to the controls for TR1 and (W - WG - PWG). We may see light bands corresponding with the reagents. Negative Result: We should not see any other dark bands. This indicates that the Walker Goal is incapable of stripping the walker off of Track 1.