Biomod/2012/UTokyo/UT-Komaba/Experiment/Lab Notes: Difference between revisions

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==October 15th==
==October 15th==


===DNA origami===
===modified DNA origami===
[[Image:Biomod-2012-UTkyo-UTKomaba-AFM-mod_origami.png]]
[[Image:Biomod-2012-UTkyo-UTKomaba-AFM-mod_origami.png]]


Revision as of 17:26, 21 October 2012

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Lab Notes

We conducted a lot of experiments at Komaba Research Campus.

September 12th

DNA Origami

We made a solution of DNA origami.

(µL)

M13 Staple Mix TAE 10x Mg2+ mQ Total Amount
A 3.00 7.20 1.80 2.25 3.75 20.00
B 3.00 3.60 1.80 2.25 7.35 20.00
C 3.00 10.80 1.80 2.25 0.15 20.00


September 13th

DNA Origami

We observed DNA origami which we made September 12th by AFM.


  • AFM image of A


  • AFM image of C


September 19th

DNA Origami

We prepared two types of DNA origami solution. The purpose of the experiment was to compare the different result from the different concentration of the solutions.

(µL)

Staple Mix M13 TAE 10x Mg2+ mQ Total Amount
A 8.00 3.33 2.00 0.25 6.42 20.00
B 12.00 5.00 2.00 0.25 0.75 20.00


DNA Origami with Enzymes

We use enzymes in bistable system, therefore we make sure that the enzymes do not destroy the DNA origami. We put nickase, polymerase and exnuclease with DNA origami and left them in the PCR. The concentration of original DNA origami was 10x and 15x.

(µL)

BST NBI tt-RecJ DTT BSA Smix DNA origami mQ Total Amount
DNA origami 10x 0.20 0.80 0.30 0.20 0.20 5.00 10.00 3.30 20.00
DNA origami 15x 0.20 0.80 0.30 0.20 0.20 5.00 10.00 3.30 20.00


DNA Origami with BSA

We are also worried if BSA interrupt the AFM and we can not get clear images of DNA origami. Therefore, we prepare the solution for September 20th. The concentration of original DNA origami was 10x and 15x.

(µL)

DNA origami Smix 4x BSA mQ Total Amount
DNA origami 10x 2.00 5.00 2.00 11.00 20.00
DNA origami 15x 2.00 5.00 2.00 11.00 20.00


Bistable System

We wanted to know the best concentration for the bistable system. Two different DNA strands, "XII" and "VII", were used for the experiment. We searched the best concentration of CvII and CxII separately.
CvII is the template for doubling V, and CxII is the one of X. "X to inhV" is the template for making inhV DNA from X, and "V to inhX" is the one of V. We change the concentration of CvII and CxII in order to find out the best concentration of them. We keep PCR in 42°C.

  • The Experiment of VII

(µL)

Bst NBI tt-RecJ DTT BSA Smix 4x V to inhX X to inhV dTTP VII CvII mQ Total Amount
5x 0.21 0.84 0.32 0.21 0.21 5.25 0.42 0.42 0.21 0.11 0.11 12.69 21.00
10x 0.21 0.84 0.32 0.21 0.21 5.25 0.42 0.42 0.21 0.11 0.21 12.59 21.00
15x 0.21 0.84 0.32 0.21 0.21 5.25 0.42 0.42 0.21 0.11 0.32 12.48 21.00
20x 0.21 0.84 0.32 0.21 0.21 5.25 0.42 0.42 0.21 0.11 0.43 12.37 21.00


  • The Experiment of XII

(µL)

Bst NBI tt-RecJ DTT BSA Smix 4x V to inhX X to inhV dTTP XII CxII mQ Total Amount
5x 0.21 0.84 0.32 0.21 0.21 5.25 0.42 0.42 0.21 0.11 0.11 12.69 21.00
10x 0.21 0.84 0.32 0.21 0.21 5.25 0.42 0.42 0.21 0.11 0.21 12.59 21.00
15x 0.21 0.84 0.32 0.21 0.21 5.25 0.42 0.42 0.21 0.11 0.32 12.48 21.00
20x 0.21 0.84 0.32 0.21 0.21 5.25 0.42 0.42 0.21 0.11 0.43 12.37 21.00


When several hours passed after keeping these tubes in 42°C, we injected XII 0.40µL in each VII tubes and VII 0.40µL in each XII tubes. Then we kept these tubes in 42°C for about 1 day.


September 20th

DNA origami

September 24th

Bistable System

We change the condition of the bistable experiment and put some tubes in PCR. The concentration of Bst and X to inhV were different from the same experiment of September 19th. We kept these tubes in 42°C.

  • The Experiment of VII

(µL)

Bst NBI tt-RecJ dTTP BSA Smix 4x V to inhX X to inhV DTTP VII CvII mQ Total Amount
5x 0.17 0.84 0.32 0.21 0.21 5.25 0.42 0.32 0.21 0.11 0.11 12.83 21.00
10x 0.17 0.84 0.32 0.21 0.21 5.25 0.42 0.32 0.21 0.11 0.21 12.73 21.00
15x 0.17 0.84 0.32 0.21 0.21 5.25 0.42 0.32 0.21 0.11 0.32 12.62 21.00
20x 0.17 0.84 0.32 0.21 0.21 5.25 0.42 0.32 0.21 0.11 0.43 12.51 21.00


  • The Experiment of XII

(µL)

Bst NBI tt-RecJ DTT BSA Smix 4x V to inhX X to inhV dTTP XII CxII mQ Total Amount
5x 0.17 0.84 0.32 0.21 0.21 5.25 0.42 0.32 0.21 0.11 0.11 12.83 21.00
10x 0.17 0.84 0.32 0.21 0.21 5.25 0.42 0.32 0.21 0.11 0.21 12.73 21.00
15x 0.17 0.84 0.32 0.21 0.21 5.25 0.42 0.32 0.21 0.11 0.32 12.62 21.00
20x 0.17 0.84 0.32 0.21 0.21 5.25 0.42 0.32 0.21 0.11 0.43 12.51 21.00


When several hours had passed since we put them in PCR, we injected XII 0.40µL in each VII tubes and VII 0.40µL in each XII tubes. Then we kept these tubes in 42°C for about 1 day as the bistable experiment we did before.


September 28th

Indirect Bistable System

We could not get a good result of the bistable system so that we change several part of the system. We introduce two types of DNA, "DII" and "NII", and two types of templates, "D to V" and "N to X" in the system. We change the concentration of "D to V" and "N to X", which are always the saame concentration. We also kept these tubes in 42°C.

  • The Experiment of XII

(µL)

BST NBI tt-RecJ DTT BSA Smix 4x V to inhX X to inhv dTTP XII CxII CvII D to V N to X mQ Total Amount
2.5nM 0.17 0.84 0.32 0.21 0.21 5.25 0.42 0.32 0.21 0.11 0.11 0.42 0.05 0.05 12.20 21.00
5.0nM 0.17 0.84 0.32 0.21 0.21 5.25 0.42 0.32 0.21 0.11 0.11 0.42 0.11 0.11 12.08 21.00
7.5nM 0.17 0.84 0.32 0.21 0.21 5.25 0.42 0.32 0.21 0.11 0.11 0.42 0.16 0.16 11.98 21.00
10.0nM 0.17 0.84 0.32 0.21 0.21 5.25 0.42 0.32 0.21 0.11 0.11 0.42 0.21 0.21 11.88 21.00


  • The Experiment of VII

(µL)

BST NBI tt-RecJ DTT BSA Smix 4x V to inhX X to inhv dTTP VII CxII CvII D to V N to X mQ Total Amount
2.5nM 0.17 0.84 0.32 0.21 0.21 5.25 0.42 0.32 0.21 0.11 0.11 0.42 0.05 0.05 12.20 21.00
5.0nM 0.17 0.84 0.32 0.21 0.21 5.25 0.42 0.32 0.21 0.11 0.11 0.42 0.11 0.11 12.08 21.00
7.5nM 0.17 0.84 0.32 0.21 0.21 5.25 0.42 0.32 0.21 0.11 0.11 0.42 0.16 0.16 11.98 21.00
10.0nM 0.17 0.84 0.32 0.21 0.21 5.25 0.42 0.32 0.21 0.11 0.11 0.42 0.21 0.21 11.88 21.00


When several hours had passed since we put these tubes in PCR, we put DII in tubes of X and NII in that of V so that the concentration of X or V might not change radically. After putting DNAs, we kept them in PCR.


October 1st

Indirect Bistable System

We conducted the same experiment as what we did in September 28th. We got enough information about the experiment of VII, so we only conducted that of VII. The purpose of the experiment is to find out the best condition of the experiment of XII. We changed the concentration of CvII and X to inhV and compared the results.

  • The Concentration of X to inhV: 10.00nM.

(µL)

BST NBI tt-RecJ DTT BSA Smix 4x V to inhX X to inhv dTTP XII CxII CvII D to V N to X mQ Total Amount
CvII: 20.00nM 0.16 0.80 0.30 0.20 0.20 5.00 0.40 0.20 0.20 0.10 0.10 0.40 0.20 0.20 11.54 20.00
CvII: 22.50nM 0.16 0.80 0.30 0.20 0.20 5.00 0.40 0.20 0.20 0.10 0.10 0.45 0.20 0.20 11.49 20.00
CvII: 25.00nM 0.16 0.80 0.30 0.20 0.20 5.00 0.40 0.20 0.20 0.10 0.10 0.50 0.20 0.20 11.44 20.00


  • The Concentration of X to inhV: 15.00nM

(µL)

BST NBI tt-RecJ DTT BSA Smix 4x V to inhX X to inhv dTTP XII CxII CvII D to V N to X mQ Total Amount
CvII: 20.00nM 0.16 0.80 0.30 0.20 0.20 5.00 0.40 0.30 0.20 0.10 0.10 0.40 0.20 0.20 11.44 20.00
CvII: 22.50nM 0.16 0.80 0.30 0.20 0.20 5.00 0.40 0.30 0.20 0.10 0.10 0.45 0.20 0.20 11.39 20.00
CvII: 25.00nM 0.16 0.80 0.30 0.20 0.20 5.00 0.40 0.30 0.20 0.10 0.10 0.50 0.20 0.20 11.34 20.00


October 2nd

Bistable System

We lowered the concentratinons of D to V, and N to X. Today, we conducted experiments in order to find out the ideal concentration of "X to inhV". We kept these tubes in 42°C, and injected once each wells.

  • The Experiment of XII

(µL)

BST NBI tt-RecJ DTT BSA Smix 4x V to inhX X to inhv dTTP XII CxII CvII mQ Total Amount
0.17 0.84 0.32 0.21 0.21 5.25 0.42 0.21 0.21 0.11 0.11 0.42 12.54 21
0.17 0.84 0.32 0.21 0.21 5.25 0.42 0.25 0.21 0.11 0.11 0.42 12.50 21
0.17 0.84 0.32 0.21 0.21 5.25 0.42 0.28 0.21 0.11 0.11 0.42 12.47 21
0.17 0.84 0.32 0.21 0.21 5.25 0.42 0.32 0.21 0.11 0.11 0.42 12.43 21

injection: VII 0.6µL each


  • The Experiment of VII

(µL)

BST NBI tt-RecJ DTT BSA Smix 4x V to inhX X to inhv dTTP VII CxII CvII mQ Total Amount
0.17 0.84 0.32 0.21 0.21 5.25 0.42 0.21 0.21 0.11 0.11 0.42 12.54 21
0.17 0.84 0.32 0.21 0.21 5.25 0.42 0.25 0.21 0.11 0.11 0.42 12.50 21
0.17 0.84 0.32 0.21 0.21 5.25 0.42 0.28 0.21 0.11 0.11 0.42 12.47 21
0.17 0.84 0.32 0.21 0.21 5.25 0.42 0.32 0.21 0.11 0.11 0.42 12.43 21

injection: XII 0.6µL each


Trioscillate System

To investigate ideal condition of trioscillate system, we conducted experiments which checks three parts of this system.

(µL)

BST NBI tt-RecJ DTT BSA Smix 4x inhmix dTTP XII CxII mQ Total Amount
0.16 0.80 0.30 0.20 0.20 5.00 0.50 0.20 0.10 0.10 12.44 20.00
0.16 0.80 0.30 0.20 0.20 5.00 0.50 0.20 0.10 0.20 12.34 20.00
0.16 0.80 0.30 0.20 0.20 5.00 0.50 0.20 0.10 0.30 12.24 20.00
0.16 0.80 0.30 0.20 0.20 5.00 0.50 0.20 0.10 0.40 12.14 20.00

injection: VII 0.80µL each


(µL)

BST NBI tt-RecJ DTT BSA Smix 4x inhmix dTTP VII CvII mQ Total Amount
0.16 0.80 0.30 0.20 0.20 5.00 0.50 0.20 0.10 0.10 12.44 20.00
0.16 0.80 0.30 0.20 0.20 5.00 0.50 0.20 0.10 0.20 12.34 20.00
0.16 0.80 0.30 0.20 0.20 5.00 0.50 0.20 0.10 0.30 12.24 20.00
0.16 0.80 0.30 0.20 0.20 5.00 0.50 0.20 0.10 0.40 12.14 20.00

injection: QII 0.80µL each


(µL)

BST NBI tt-RecJ DTT BSA Smix 4x inhmix dTTP QII CqII mQ Total Amount
0.16 0.80 0.30 0.20 0.20 5.00 0.50 0.20 0.10 0.10 12.44 20.00
0.16 0.80 0.30 0.20 0.20 5.00 0.50 0.20 0.10 0.20 12.34 20.00
0.16 0.80 0.30 0.20 0.20 5.00 0.50 0.20 0.10 0.30 12.24 20.00
0.16 0.80 0.30 0.20 0.20 5.00 0.50 0.20 0.10 0.40 12.14 20.00

injection: XII 0.80µL each


October 3rd

Bistable System

In today's experiment, we investigated the ideal temperature for the bistable system. We made two solutions (one starts from "X", and the other from "Y"), and observed at 6 different temperatures - 41.6～47.6℃.

(µL)

BST NBI tt-RecJ DTT BSA Smix 4x D to V,N to X V to inhX X to inhv dTTP XII VII CxII CvII mQ Total Amount
0.16 0.80 0.30 0.20 0.20 5.00 1.50 0.40 0.30 0.20 0.10 0.00 0.10 0.40 10.34 20.00
0.16 0.80 0.30 0.20 0.20 5.00 1.50 0.40 0.30 0.20 0.00 0.10 0.10 0.40 10.34 20.00

injection: XII or VII 0.60µL each

October 15th

modified DNA origami

October 11th

Trioscillate system

Today, we tested whole trioscillate system. To investigate the ideal condition, we tested both direction of inhibition (V-|X-|Q, V-|Q-|X) and each concentration of CxII, CvII, CqII.


  • inhibit direction: V-|X-|Q

(µL)

BST NBI tt-RecJ DTT BSA Smix 4x inhmix dTTP XII VII CxII CvII CqII mQ Total Amount
0.16 0.80 0.30 0.20 0.20 5.00 0.40 0.10 0.11 0.11 0.50 0.40 0.50 11.22 20.00
0.16 0.80 0.30 0.20 0.20 5.00 0.40 0.10 0.11 0.11 0.50 0.40 1.00 10.72 20.00
0.16 0.80 0.30 0.20 0.20 5.00 0.40 0.10 0.11 0.11 0.50 0.40 1.50 10.22 20.00
0.16 0.80 0.30 0.20 0.20 5.00 0.40 0.10 0.11 0.11 0.50 0.40 2.00 9.72 20.00
0.16 0.80 0.30 0.20 0.20 5.00 0.40 0.10 0.11 0.11 1.00 0.40 0.50 10.72 20.00
0.16 0.80 0.30 0.20 0.20 5.00 0.40 0.10 0.11 0.11 1.00 0.40 1.00 10.22 20.00
0.16 0.80 0.30 0.20 0.20 5.00 0.40 0.10 0.11 0.11 1.00 0.40 1.50 9.72 20.00
0.16 0.80 0.30 0.20 0.20 5.00 0.40 0.10 0.11 0.11 1.00 0.40 2.00 8.22 20.00
0.16 0.80 0.30 0.20 0.20 5.00 0.40 0.10 0.11 0.11 1.50 0.40 0.50 10.22 20.00
0.16 0.80 0.30 0.20 0.20 5.00 0.40 0.10 0.11 0.11 1.50 0.40 1.00 9.72 20.00
0.16 0.80 0.30 0.20 0.20 5.00 0.40 0.10 0.11 0.11 1.50 0.40 1.50 9.22 20.00
0.16 0.80 0.30 0.20 0.20 5.00 0.40 0.10 0.11 0.11 1.50 0.40 2.00 8.72 20.00
0.16 0.80 0.30 0.20 0.20 5.00 0.40 0.10 0.11 0.11 2.00 0.40 0.50 9.72 20.00
0.16 0.80 0.30 0.20 0.20 5.00 0.40 0.10 0.11 0.11 2.00 0.40 1.00 9.22 20.00
0.16 0.80 0.30 0.20 0.20 5.00 0.40 0.10 0.11 0.11 2.00 0.40 1.50 8.72 20.00
0.16 0.80 0.30 0.20 0.20 5.00 0.40 0.10 0.11 0.11 2.00 0.40 2.00 8.22 20.00


  • inhibit direction: V-|Q-|X

(µL)

BST NBI tt-RecJ DTT BSA Smix 4x inhmix dTTP XII VII CxII CvII CqII mQ Total Amount
0.16 0.80 0.30 0.20 0.20 5.00 0.40 0.10 0.11 0.11 0.10 0.50 0.50 11.52 20.00
0.16 0.80 0.30 0.20 0.20 5.00 0.40 0.10 0.11 0.11 0.10 0.50 1.00 11.02 20.00
0.16 0.80 0.30 0.20 0.20 5.00 0.40 0.10 0.11 0.11 0.10 0.50 1.50 10.52 20.00
0.16 0.80 0.30 0.20 0.20 5.00 0.40 0.10 0.11 0.11 0.10 0.50 2.00 10.02 20.00
0.16 0.80 0.30 0.20 0.20 5.00 0.40 0.10 0.11 0.11 0.10 1.00 0.50 11.02 20.00
0.16 0.80 0.30 0.20 0.20 5.00 0.40 0.10 0.11 0.11 0.10 1.00 1.00 10.52 20.00
0.16 0.80 0.30 0.20 0.20 5.00 0.40 0.10 0.11 0.11 0.10 1.00 1.50 10.02 20.00
0.16 0.80 0.30 0.20 0.20 5.00 0.40 0.10 0.11 0.11 0.10 1.00 2.00 9.52 20.00
0.16 0.80 0.30 0.20 0.20 5.00 0.40 0.10 0.11 0.11 0.10 1.50 0.50 10.52 20.00
0.16 0.80 0.30 0.20 0.20 5.00 0.40 0.10 0.11 0.11 0.10 1.50 1.00 10.02 20.00
0.16 0.80 0.30 0.20 0.20 5.00 0.40 0.10 0.11 0.11 0.10 1.50 1.50 9.52 20.00
0.16 0.80 0.30 0.20 0.20 5.00 0.40 0.10 0.11 0.11 0.10 1.50 2.00 9.02 20.00
0.16 0.80 0.30 0.20 0.20 5.00 0.40 0.10 0.11 0.11 0.10 2.00 0.50 10.02 20.00
0.16 0.80 0.30 0.20 0.20 5.00 0.40 0.10 0.11 0.11 0.10 2.00 1.00 9.52 20.00
0.16 0.80 0.30 0.20 0.20 5.00 0.40 0.10 0.11 0.11 0.10 2.00 1.50 9.02 20.00
0.16 0.80 0.30 0.20 0.20 5.00 0.40 0.10 0.11 0.11 0.10 2.00 2.00 8.52 20.00


October 12th

Trioscillate system

We tested again the wells which worked well in before experiment.

  • inhibit direction: V-|X-|Q

(µL)

BST NBI tt-RecJ DTT BSA Smix 4x inhmix dTTP XII VII CxII CvII CqII mQ Total Amount
0.16 0.80 0.30 0.20 0.20 5.00 0.40 0.10 0.11 0.11 0.50 0.40 0.50 11.22 20.00
0.16 0.80 0.30 0.20 0.20 5.00 0.40 0.10 0.11 0.11 0.50 0.40 1.00 10.72 20.00
0.16 0.80 0.30 0.20 0.20 5.00 0.40 0.10 0.11 0.11 0.50 0.40 1.50 10.22 20.00
0.16 0.80 0.30 0.20 0.20 5.00 0.40 0.10 0.11 0.11 0.50 0.40 2.00 9.72 20.00
0.16 0.80 0.30 0.20 0.20 5.00 0.40 0.10 0.11 0.11 1.00 0.40 0.50 10.72 20.00
0.16 0.80 0.30 0.20 0.20 5.00 0.40 0.10 0.11 0.11 1.00 0.40 1.00 10.22 20.00
0.16 0.80 0.30 0.20 0.20 5.00 0.40 0.10 0.11 0.11 1.00 0.40 1.50 9.72 20.00
0.16 0.80 0.30 0.20 0.20 5.00 0.40 0.10 0.11 0.11 1.00 0.40 2.00 8.22 20.00
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