User:Allison K. Alix/Notebook/Thesis Research/2013/07/25

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(ThT/DNA attachment)
Current revision (15:12, 25 July 2013) (view source)
(ThT/DNA attachment)
 
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1) Dilute 23.5nM AuNPs to 9.4nM (2mL AuNPs in 3mL water *NOTE: the water was left to assimilate to room temperature prior to dilution*)
1) Dilute 23.5nM AuNPs to 9.4nM (2mL AuNPs in 3mL water *NOTE: the water was left to assimilate to room temperature prior to dilution*)
-
2)  
+
2) Prepare 5mL 10mM MHA in water and isopropanol
 +
 
 +
3) Mix 5mL AuNPs w/ 5mL MHA
 +
 
 +
4) Stir overnight
==ThT/DNA attachment==
==ThT/DNA attachment==
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e) 900nM
e) 900nM
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***Original Concentrations of 4 and 8-repeats are 265.6uM and 142.8uM, respectively***
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NOTE: Original Concentrations of 4 and 8-repeats are 265.6uM and 142.8uM, respectively
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4-repeat
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'''4-repeat'''
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x(500uL)=(265.6uM)(8.12)
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x(500uL)=(265.6uM)(10)
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x = 4.31uM
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x = 5.31uM
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1800nM-->  41.8uL in 58.2uL PBS
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1800nM-->  33.9uL in 66.1uL PBS  
-
1400nM--> 32.5uL in 67.5uL
+
1400nM--> 26.4uL in 73.6uL
-
1000nM--> 23.2uL in 76.8uL
+
1000nM--> 18.8uL in 81.2uL
-
600nM--> 13.9uL in 86.1uL
+
600nM--> 11.3uL in 88.7uL
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200nM--> 4.6uL in 95.4uL
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200nM--> 3.8uL in 96.2uL
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8-repeat
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'''8-repeat'''
142.8uM(10uL) = (x)(500uL)
142.8uM(10uL) = (x)(500uL)
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x= 2.856
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x= 2.856uM
1800nM-->  63uL in 37uL PBS
1800nM-->  63uL in 37uL PBS
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200nM--> 7uL in 93uL
200nM--> 7uL in 93uL
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 +
* excite at 417nm and run from 427-727 w/ 10nm slit width. Maximum peak @ ~490nm.
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 +
==Fluorescence Measurements==
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 +
[[Image:OWW_pic_4_repeat.png]]
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[[Image:OWW_pic_8_repeat.png]]
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[[Image:OWW_pic_cali_curve.png]]
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==Observations==
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 +
The graphs above show the following: the fluorescence intensity of varying concentrations of 4-repeat telomeric DNA with 2.5uM ThT, the fluorescence intensity of varying concentrations of 8-repeat telomeric DNA (the same as the 4-repeat) with 2.5uM ThT, and a calibration curve displaying fluorescence intensity vs. concentration for the two different strands. From the calibration curve, two things are obvious: 1) there is an increase in fluorescence with an increase in DNA concentration independent of the the length of the strand (which we have seen before with the S-DNA strand) and 2) There is an increase in fluorescence when concentration is held constant and the number of telomeric repeats (TTAGGG) is increased. This is important as it is now evident that fluorescence can be measured to distinguish between amounts of DNA present as well as lengths of strands.

Current revision

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To Do

  • Repeat Silica-Coating procedure using water to dilute nanoparticles instead of PBS. PBS is likely the cause of the aggregation we have been observing.[1]
  • Repeat ThT-DNA fluorescence procedure to show that fluorescence increases with increasing number of G-quadruplexes formed.

Silica-Coating Part 1

1) Dilute 23.5nM AuNPs to 9.4nM (2mL AuNPs in 3mL water *NOTE: the water was left to assimilate to room temperature prior to dilution*)

2) Prepare 5mL 10mM MHA in water and isopropanol

3) Mix 5mL AuNPs w/ 5mL MHA

4) Stir overnight

ThT/DNA attachment

1) Prepare 100uL of the following concentrations of 4-strand and 8-strand telomeric DNA (which should generate 1 and 2 G-quadruplexes respectively)

a) 100nM

b) 300nM

c) 500nM

d) 700nM

e) 900nM

NOTE: Original Concentrations of 4 and 8-repeats are 265.6uM and 142.8uM, respectively

4-repeat

x(500uL)=(265.6uM)(10)

x = 5.31uM

1800nM--> 33.9uL in 66.1uL PBS

1400nM--> 26.4uL in 73.6uL

1000nM--> 18.8uL in 81.2uL

600nM--> 11.3uL in 88.7uL

200nM--> 3.8uL in 96.2uL

8-repeat

142.8uM(10uL) = (x)(500uL)

x= 2.856uM

1800nM--> 63uL in 37uL PBS

1400nM--> 49uL in 51uL

1000nM--> 35uL in 65uL

600nM--> 21uL in 79uL

200nM--> 7uL in 93uL

  • excite at 417nm and run from 427-727 w/ 10nm slit width. Maximum peak @ ~490nm.

Fluorescence Measurements

Image:OWW_pic_4_repeat.png


Image:OWW_pic_8_repeat.png


Image:OWW_pic_cali_curve.png

Observations

The graphs above show the following: the fluorescence intensity of varying concentrations of 4-repeat telomeric DNA with 2.5uM ThT, the fluorescence intensity of varying concentrations of 8-repeat telomeric DNA (the same as the 4-repeat) with 2.5uM ThT, and a calibration curve displaying fluorescence intensity vs. concentration for the two different strands. From the calibration curve, two things are obvious: 1) there is an increase in fluorescence with an increase in DNA concentration independent of the the length of the strand (which we have seen before with the S-DNA strand) and 2) There is an increase in fluorescence when concentration is held constant and the number of telomeric repeats (TTAGGG) is increased. This is important as it is now evident that fluorescence can be measured to distinguish between amounts of DNA present as well as lengths of strands.






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