Difference between revisions of "Team:SEU/Experiments"

Experiments

To demonstrate our calculations simulated by computer, we carried out following experiments. We will show these experiments in chronological order and present the whole process of our trial and error and improvements in experimental method.

All the experiments can be categorized into six parts:

a. Acquire single chain DNA sequences used in wet experiments (we got these sequences by our Software Tool).
b. Synthesize DNA in part 1 (in fact we bought them from Sangon Biotech).
c. Dissolve, dilute, mix.
d. Denature and anneal (acquire the results of fluorescence intensity by qRT-PCR simultaneously).
e. Purify and quantify the reactant complexes.
f. Characterization via PAGE

Name	Sequence	Length
1ai	ACACCATCCATCCACACCATACTACCTCTCTCCACCATAC	40
1Gia	ACTACCTCTCTCCACCATACACCACACCACCACACA CCATACCTCACCACTCCACACTCT	60		3`6-FAM
1Gib	TGGTATGATGGAGAGAGGTGGTATG	25
1Tia	ACCACACCACCACACACCATACTCTCTCCTTCCACCATAC	40
1Tib	ACCTCACCACTCCACACTCTACCTCCACAACCAACTCTAC	40
1Tic	TGGTATGGTGTGGTGGTGTGTGGTATGGAGTGGTGAGGTGTGAGA	45	5`BHQ1
2ai	ACACCATCCATCCACACTCAACTACCTCTCTCCACTCAAC	40
2Lia1	ACTACCTCTCTCCACTCAACACATC	25
2Lia2	ACTCTCTCCTTCCACATCACACCTCACCACTCCACACTCA	40		3`6-FAM
2Lib	TGAGTTGATGGAGAGAGGTGAGTTGTGTAGTGAGAGAGGAAGGTGTAGTG	50
2bi	ACCACACCACCACACACATCACTCTCTCCTTCCACATCAC	40
2Tia	ACCTCACCACTCCACACTCAACCTCCACAACCAACTCAAC	40
2Tib	TAGTGTGGAGTGGTGAGGTGTGAGT	25	5`BHQ1
3ai	ACCTCCTCCTCCTACACAACACCACCTCCTCCAACAACAC	40
3Lia1	ACCACCTCCTCCAACAACACACTCT	25
3Lia2	ACACCTTCTTTACACTCTACACCTCCTCC TCCTACACAACACCTTTCCTCCTCACACTCTACCTCCTCACCTCACACTTC	80		3`6-FAM
3Lib	TGTTGTGGTGGAGGAGGTTGTTGTGTGAGATGTGGAAGAAATGTGAGATG	50
3b	ACCTTTCCTCCTCACACTCTACACCTTCTTTACACTCTAC	40
3Tic	ACCTCCTCACCTCACACTTCACACCTTCTTTACACTCTAC	40
3Tid	AGATGTGGAGGAGGAGGATGTGTTGTGGAAAGGAGGA GTGTGAGATGGAGGAGTGGAGTGTGAAG	65	5`BHQ1
3Wia	ACCACCTCCTCCAACAACAC	20
3Wib	GTGTTGTTGGAGGAGGTGGTGTTGT	25

Basic experiment details are as follows:

1. Dissolve, dilute, mix

All the single chain DNAs were dissolved in DEPC-treated water to form 100 uM solutions respectively and saved at 4 ℃.
Reactant complexes were annealed together at 20 uM in Tris-acetate-EDTA buffer containing 12.5 mM Mg2+(1×TAE/Mg2+) and saved at 4 ℃ for further experiments.

2. Gel electrophoresis

To examine the products of every kinetics experiments and purify the reactant complexes, 12% non-denaturing PAGE was run at 40 V for about 3.5 hours. After staining with GelRed (Biotium, 1:10000) for 30 min, the gel was scanned on a gel imaging system (Tanon 3500R).
Formulation for 12% native PAGE is as follows:

29:1 30% Acrylamide/bis	Deionized Water	5xTBE Buffer	10% APS	TEMED	Total Volume
4mL	3.93mL	2mL	0.07mL	0.007mL	10mL

We use both double stranded DNA (dsDNA) ladder marker and single stranded DNA (ssDNA) ladder marker in gel electrophoresis. Others like reactants were also utilized as markers because DNAs in our reaction systems included both ssDNA and dsDNA, especially with long sticky ends or cohesive ends, which was hard to recognize and analyse though mere dsDNA ladder marker and ssDNA ladder marker.

3. Reactant complexes purification and quantification

After acquiring the reactant complexes, we used them to carry out the polyacrylamide gel electrophoresis and obtained the target stripes cut down from the gel with the assistance of a gel imaging system (Tanon 3500R). Gel fragments were broken up and suspended in TE buffer about the same volume as the fragments and kept in 37 ℃ for 12 hours to release the DNAs. Then we obtained the DNA solutions after centrifugation (15000 rpm, 5 min) and used another 0.5 time of volume of TE buffer to get the remaining DNAs by repeating the process mentioned above. After that, ethanol at 4 ℃ as much as two folds of the extraction solution was added in and then the mixtures were put on ice for 30 min. The mixtures were centrifuged at 15000 rpm for 20 min to get the DNA precipitate. Afterwards, 200 uL TE buffer (pH 8.0) was added to dissolve DNAs and then 25 uL 3 mol/L NaAc (pH 5.2) was added. Another 450 uL ethanol and 1 uL 20 mg/mL glycogen were added into the mixtures to re-precipitate the DNAs. After 12 hours, the mixtures were centrifuged at 15000 rpm for 20 min and washed with 70 % (V/V) ethanol, then we used 20 uL TE buffer (pH 8.0) to dissolve the precipitate to get the purified reactant complexes. XXXXXXXX(nanodrop的型号) was used to quantify the concentrations of DNAs of these products.

4. Kinetics experiments

Denaturing and annealing were performed in a quantitative real-time PCR (qRT-PCR) machine, first heating up to 95 oC, then slowly cooling down to 20 ℃ at the rate of 1 ℃/min, and finally holding at 20 ℃ for 2－4 hours.
For the same sample, we tried two concentrations of reactants, that is, 1 uM and 0.1 uM. For each concentration, we used three reaction cups to carry out the experiments, 20 uL in each cup.
Reactant complexes were synthesized with the reactant amount at 20 uM.

Experiment process are as follows:

1. Synthesis of reactant complexes

Reactant complexes were annealed together at 20 uM in Tris-acetate-EDTA buffer containing 12.5 mM Mg2+(1×TAE/Mg2+) and saved at 4 ℃ for further experiments. Volumes for all the reaction system here at one time are all 50 uL. The concrete details are shown in the following table.

1Gi (Reactant Complex for Addition)
1Gia (100 uM)	10uL
1Gib (100 uM)	10uL
Deionized Water	25uL
10×TAE Buffer	5uL

1Ti (Reactant Complex for Addition)
1Tia (100 uM)	10uL
1Tib (100 uM)	10uL
1Tic (100 uM)	10uL
Deionized Water	15uL
10×TAE Buffer	5uL

2Li (Reactant Complex for Addition)
2Lia1 (100 uM)	10uL
2Lia2 (100 uM)	10uL
2Lib (100 uM)	10uL
Deionized Water	15uL
10×TAE Buffer	5uL

2Ti (Reactant Complex for Addition)
1Tia (100 uM)	10uL
1Tib (100 uM)	10uL
Deionized Water	25uL
10×TAE Buffer	5uL

3Li (Reactant Complex for Addition)
3Lia1 (100 uM)	10uL
3Lia2 (100 uM)	10uL
3Lib (100 uM)	10uL
Deionized Water	15uL
10×TAE Buffer	5uL

3Ti (Reactant Complex for Addition)
3Tic (100 uM)	10uL
3Tid (100 uM)	10uL
3a (100 uM)	10uL
3b (100 uM)	10uL
Deionized Water	5uL
10×TAE Buffer	5uL

3Wi (Reactant Complex for Addition)
3WiUp (100 uM)	10uL
3WiDown (100 uM)	10uL
Deionized Water	25uL
10×TAE Buffer	5uL

2. Kinetics experiments of addition

After we obtained the reactant complexes, we began to use these DNAs to study their kinetic feature. We carried out experiments one after another using different experimental conditions, including using reactant complexes before or after purification, one-step or multi-step and different types of quantitative real-time PCR (qRT-PCR) machine.
Firstly, we wanted to study whether denaturing and annealing step by step is more valid than just mixing and running in one time when testing a kind of calculation, so we kept other conditions unchanged, all using unrefined reactant complexes and the same PCR machine (BIO-RAD CFX96). Then we found there are not obvious difference between these two kinds of strategies. The detailed compositions of every reaction system are shown in the table below. Every reaction system were designed to contain 70 uL and then divided into three reaction cups with 20 uL a cup.

		10uM (Oi)	1uM	0.1uM
One-Step	1ai (100uM)		0.7uL	0.07uL
	1Gi (20uM)		3.5uL	0.35uL
	1Ti (20uM)		3.5uL	0.35uL
	10x Buffer
	Deionized Water		56uL	61.67uL

However, it occurred that the results are somewhat odd in that the fluorescence intensities of some circles are negative despite that we repeated these experiments for several times, which means that something must be wrong with the experiments. And, although with some strange points, the general tendency was right because we could see a sudden decrease of fluorescence intensity during annealing, which could not happen without our target single stranded DNAs combining and fluorophores encountering quenchers.
In order to demonstrate the conduct of the addition reaction, we used 12% native PAGE to visualize the DNA fragments. And we could observe the formation of Oi (the target product of the first step of two-step group) and the mixture of b and c (the target product of the whole experiment). It is because b and c share the same length XXXXXXXXXXXX
To reduce the interference of unreacted single stranded DNAs in reactant complexes, we conducted purification to the reactant complexes (1Gi and 1Ti in addition reaction) after above experiments.
As for the odd data points referred above, we tried three types of qRT-PCR machine, that is, BIO-RAD CFX96, Applied Biosystems QuantStudio 3 and Applied Biosystems StepOnePlus Real-Time PCR Systems. Results are shown as follows:

3. Kinetics experiments of subtraction

Just like what we did in addition reaction part, we also tried different strategies and different machines.

4. Kinetics experiments of multiplication

Different from addition and subtraction, there is a kind of short double-stranded DNA called exponential factor in multiplication reaction. How we utilize them is to change the concentration or ratio of them and to see whether it make a difference in the reaction system.