User:Pranav Rathi/Notebook/OT/2013/01/09/DNA-Overstretching Experiments: Difference between revisions
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== Experiment 6 (01/15/2013)== | |||
I did two overstretching experiments today with H2O and D2O with 4.4kb (110mg/ml;02/11/11) DNA. From tomorrow i will be using new DNA so i think this is the last experiment i have with this DNA, since it is getting old now. | |||
Overstretching in H2O and D2O worked fine but D2O was lot better than H2O. | |||
===Experiment:1=== | |||
Overstrething in H2O | |||
====Sample preparation==== | |||
Single-chamber sample. | |||
'''Bead:''' | |||
*H2O: | |||
1.5ul of 265nm bead (1:5) from stock+13.5ul of BGB H2O=15ul of bead H2O (1:50) | |||
'''DNA:''' | |||
ds-DNA 4.4kb (110ng/ml;02/11/11) | |||
*D2O; | |||
1ul of DNA from stock (1:10) + 9ul of 1XPOP H2O =10ul of DNA H2O (1:100) | |||
===Procedure=== | |||
* This procedure is working great so I am going to use the same procedure for all the future sample I will make with 265nm beads. | |||
# Flow anti-dig 12 ul wat for 6min; D2O | |||
# Flow BGB 50ul twice, wait for 2 min; D2O | |||
# Flow DNA 10 ul, wait for 12 min; D2O | |||
# Sonicate beads 90sec | |||
# Flow BGB 50ul twice, wait none; D2O | |||
# Flow beads, wait for 12 min; D2O | |||
# Flow BGB 50 ul-Thrice, wait none; | |||
# Seal it | |||
===Result:=== | |||
Revision as of 11:51, 16 January 2013
Introduction
This Page contains all the information regarding DNA-Overstretching experiments and results.
{{#widget:Picasa |user=101108414393941264686 |album=5831510904997812145 |width=600 |height=400 |captions=1 |autoplay=0 }}
Proof of DNA-tethering
Two video presents good DNA-tethers in H2O and D2O. For some reason D2O tethers look little better. Each moving bead has a ds-DNA tether which is roughly 1500nm long (4.4kb; 110ng/ml; 02/11/11). The bead size is 530nm diameter. This sample is prepared in water and heavy water for DNA-overstretching experiments which i will discuss below. {{#widget:YouTube|id=vKV4CZYMXSw}}{{#widget:YouTube|id=ihl45JssNTA}}
Experiments
Experiment 1 (01/04/2013)
Prepare some new buffers and attempt DNA-tethering in H2O and D2O with 265nm beads (bead radius).
Buffer preparation
BGB is good only for few weeks and it has been over 2 months since i made it last, so i am going to make new BGB and antidig. For more information go to Buffer preparation for DNA overstretching and unzippingexperiments[1]
BGB
- H2O
50mg of BGB + 10ml of H2O 1X POP=5mg/ml BGB 1XPOP H2O 10ml
- D2O
50mg of BGB + 10ml of D2O 1X POP=5mg/ml BGB 1XPOP D2O 10ml
Antidig
Same for both H2O and D2O since PBS is in H2O only.
20ul of aliquots + 180ul of PBS H2O=200ul of antidig H2O
Sample preparation
Dual-chamber sample. For more information go to sample preparation for DNA overstretching & unzipping experiments: [2]
Bead:
- H2O:
1.5ul of 265nm bead (1:5) from stock+13.5ul of BGB H2O=15ul of bead H2O (1:5o)
- D2O:
1.5ul of 265nm bead (1:5) from stock+13.5ul of BGB D2O=15ul of bead D2O (1:5o)
DNA: ds-DNA 4.4kb (110ng/ml;02/11/11)
- H2O;
1ul of DNA from stock (1:10) + 9ul of 1XPOP H2O =10ul of DNA H2O (1:100)
- D2O;
1ul of DNA from stock (1:10) + 9ul of 1XPOP D2O =10ul of DNA D2O (1:100)
Procedure
- Flow anti-dig 12 ul wat for 6min; H2O and D2O
- Flow BGB 50ul twice, wait for 2 min; H2O and D2O
- Flow DNA 10 ul, wait for 12 min; H2O and D2O
- Sonicate beads 90sec
- Flow BGB 50ul twice, wait none; H2O and D2O
- Flow beads, wait for 12 min; H2O and D2O
- Flow 1XPOP 50 ul-Twice, wait none; H2O and D2O
- Seal it
Results:
- Lots of stuck bead most of the beads are stuck, very few tethers which overstretch just fine.
- QPD X and Y ramping problem is back.
- conclusion:
With 520nm big beads flow 1XPOP in the last step with 265nm small beads flow BGB in the last step.
Data
- Data is in file: 1301104-0621 to 0626.
- The data link at server: [3]
Data and other information can be seen through evernotes:
<html><iframe height="300px" width=600px" src="http://www.evernote.com/shard/s24/sh/5ec1bc8a-47d9-481f-b3c8-675768d959d2/8b754e9f2fa8d61316172629aabae7c6"></iframe></html>
Experiment 2 (01/05/2013)
The ramping problem is fixed by taking the ND filter away from the QPD and introducing a delay-step in data acquisition process. DNA Overstretching experiments in H2O and D2O:
Sample preparation
Dual-chamber sample.
Bead:
- H2O:
1.5ul of 265nm bead (1:5) from stock+13.5ul of BGB H2O=15ul of bead H2O (1:50)
- D2O:
1.5ul of 265nm bead (1:5) from stock+13.5ul of BGB D2O=15ul of bead D2O (1:50)
DNA: ds-DNA 4.4kb (110ng/ml;02/11/11)
- H2O;
1ul of DNA from stock (1:10) + 9ul of 1XPOP H2O =10ul of DNA H2O (1:100)
- D2O;
1ul of DNA from stock (1:10) + 9ul of 1XPOP D2O =10ul of DNA D2O (1:100)
Procedure
- Flow anti-dig 12 ul wat for 6min; H2O and D2O
- Flow BGB 50ul twice, wait for 2 min; H2O and D2O
- Flow DNA 10 ul, wait for 12 min; H2O and D2O
- Sonicate beads 90sec
- Flow BGB 50ul twice, wait none; H2O and D2O
- Flow beads, wait for 12 min; H2O and D2O
- Flow BGB 50 ul-Twice, wait none; H2O and D2O
- Seal it
Result:
- Very successful tethering and overstretching, bead start stucking more in h2o after 1 hour, so flow BGB thrice next time.
- Ready to work on find tether center (FTC)
Data
- Data is in file: 1301105-0631 to 0637
segment 0627-0634 D2O segment 0635-0637 H2O
- The data link at server:[4]
Data and other information can be seen through evernotes:
<html><iframe height="300px" width=600px" src="http://www.evernote.com/shard/s24/sh/e7884053-f51c-412b-9091-387e5bdd59e7/f5caca1d7a8bbcf9bb7104fadabdda02"></iframe></html>
Experiment 3 (01/07/2013)
FTC was fixed for big beads. Find tether center (FTC) is a step sequence program which finds a tether center, centers the trap and acquires data for repeatable geometer. This experiment I used big beads (radius 520 nm).
Sample preparation
Dual-chamber sample.
Bead:
- H2O:
1.5ul of 520nm bead (1:5) from stock+13.5ul of BGB H2O=15ul of bead H2O (1:50)
- D2O:
1.5ul of 520nm bead (1:5) from stock+13.5ul of BGB D2O=15ul of bead D2O (1:50)
DNA: ds-DNA 4.4kb (110ng/ml;02/11/11)
- H2O;
1ul of DNA from stock (1:10) + 9ul of 1XPOP H2O =10ul of DNA H2O (1:100)
- D2O;
1ul of DNA from stock (1:10) + 9ul of 1XPOP D2O =10ul of DNA D2O (1:100)
Procedure
- Flow anti-dig 12 ul wat for 6min; H2O and D2O
- Flow BGB 50ul twice, wait for 2 min; H2O and D2O
- Flow DNA 10 ul, wait for 12 min; H2O and D2O
- Sonicate beads 90sec
- Flow BGB 50ul twice, wait none; H2O and D2O
- Flow beads, wait for 12 min; H2O and D2O
- Flow BGB 50 ul-Twice, wait none; H2O and D2O
- Seal it
Result:
- Few tether, fewer than small bead sample, for big bead flow 1XPOP in the end not the BGB.
- Find tether center was fixed and worked great with this sample.
Data
- Data acquisition parameters: DNA4.4kb(110ng/ml; 02/11/11) ,r=520nm (bead radius) ,medium h2o and d20,tch/BH886nm (trap center height or bead center height from surface),fh700nm (focal height from surface). TCH/BH = trap center offset + focal height from surface (=186+700), The best focal height is 600nm which gives TCH/BH of 786 nm. This height is not too height or too low.
- Data is in file: 1301107/000 to 0016
segment 0012-0016 D2O segment 0000-0011 H2O
- The data link at server:http:
Overstretching-data and other information can be seen through evernotes:
<html><iframe height="300px" width=600px" src="http://www.evernote.com/shard/s24/sh/c7454c09-bb94-4b25-a4f9-779f94345eb8/4af871f64fc7d1b9e9b06b1f4893a294"></iframe></html>
Find tether center steps can be seen through this link:
<html><iframe height="300px" width=600px" src="http://www.evernote.com/shard/s24/sh/16ffe42f-484e-4ca8-b157-d16465c1207e/15dcf8745341f8a07ed03bbf6619f586"></iframe></html>
Experiment 4 (01/09/2013)
FTC was fixed for small beads. The overstretching is done only n H2O.
Sample preparation
Single-chamber sample.
Bead:
- H2O:
1.5ul of 265nm bead (1:5) from stock+13.5ul of BGB H2O=15ul of bead H2O (1:50)
DNA: ds-DNA 4.4kb (110ng/ml;02/11/11)
- H2O;
1ul of DNA from stock (1:10) + 9ul of 1XPOP H2O =10ul of DNA H2O (1:100)
Procedure
- Flow anti-dig 12 ul wat for 6min; H2O
- Flow BGB 50ul twice, wait for 2 min; H2O
- Flow DNA 10 ul, wait for 12 min; H2O
- Sonicate beads 90sec
- Flow BGB 50ul twice, wait none; H2O
- Flow beads, wait for 12 min; H2O
- Flow BGB 50 ul-Thrice, wait none;
- Seal it
Result:
- Sample was very successful lot of good tethers and sample was still good more than over 3 hour period.
- The overstretching data looks really good the overstretching force is at 65pN.
- FTC was fixed and working great.
Data
- Data acquisition parameters: DNA4.4kb(110ng/ml; 02/11/11) ,r=265nm ,medium h2o,TCH/BH 574nm,FH 200 & 300nm. The best focal height is 300nm which gives TCH/BH of 674 nm. But this data was taken at TCH/BH of 574nm. This height is not too height or too low.
- Data is in file: 1301109\004
- The data link at server:http:
Overstretching-data and other information can be seen through evernotes:
<html><iframe height="300px" width=600px" src="http://www.evernote.com/shard/s24/sh/af647c4a-de9d-4de3-8d82-ed4c851ad4f5/a1628d7d84b71052d05133dc35e1c57a"></iframe></html>
Find tether center steps can be seen through this link:
<html><iframe height="300px" width=600px" src="http://www.evernote.com/shard/s24/sh/91c4c24c-8978-4612-a6be-87ce300ea8bc/189fba3e1227430d8cb302a4bf212def"></iframe></html>
Experiment 5 (01/10/2013)
Overstretching in D2O. With quick conversion the data looks great and now we are trying to work geometry out and do a full length data conversion and comparison between H2O and D2O.
Sample preparation
Single-chamber sample.
Bead:
- D2O:
1.5ul of 265nm bead (1:5) from stock+13.5ul of BGB H2O=15ul of bead H2O (1:50)
DNA: ds-DNA 4.4kb (110ng/ml;02/11/11)
- D2O;
1ul of DNA from stock (1:10) + 9ul of 1XPOP H2O =10ul of DNA H2O (1:100)
Procedure
- This procedure is working great so I am going to use the same procedure for all the future sample I will make with 265nm beads.
- Flow anti-dig 12 ul wat for 6min; D2O
- Flow BGB 50ul twice, wait for 2 min; D2O
- Flow DNA 10 ul, wait for 12 min; D2O
- Sonicate beads 90sec
- Flow BGB 50ul twice, wait none; D2O
- Flow beads, wait for 12 min; D2O
- Flow BGB 50 ul-Thrice, wait none;
- Seal it
Result:
- Sample is very successful lots of good tethers.
- D2O sample looks better then H2O sample, Temperature is about the same as 76degF.
- The overstretching data looks really good the overstretching force is little higher about 68pN.
Data
- Data acquisition parameters: DNA4.4kb(110ng/ml; 02/11/11) ,r=265nm ,medium h2o,TCH/BH 574nm,FH 200 & 300nm.
- Data is in file: 1301110\001 to 003.
- Good segments are: 001-seg2,8,10,14,17,19,20,22,26,29,30,32,33,42,56,58,62,64
- The data link at server:http:
Overstretching-data and other information can be seen through evernotes:
<html><iframe height="300px" width=600px" src="http://www.evernote.com/shard/s24/sh/af647c4a-de9d-4de3-8d82-ed4c851ad4f5/a1628d7d84b71052d05133dc35e1c57a"></iframe></html>
Experiment 6 (01/15/2013)
I did two overstretching experiments today with H2O and D2O with 4.4kb (110mg/ml;02/11/11) DNA. From tomorrow i will be using new DNA so i think this is the last experiment i have with this DNA, since it is getting old now.
Overstretching in H2O and D2O worked fine but D2O was lot better than H2O.
Experiment:1
Overstrething in H2O
Sample preparation
Single-chamber sample.
Bead:
- H2O:
1.5ul of 265nm bead (1:5) from stock+13.5ul of BGB H2O=15ul of bead H2O (1:50)
DNA: ds-DNA 4.4kb (110ng/ml;02/11/11)
- D2O;
1ul of DNA from stock (1:10) + 9ul of 1XPOP H2O =10ul of DNA H2O (1:100)
Procedure
- This procedure is working great so I am going to use the same procedure for all the future sample I will make with 265nm beads.
- Flow anti-dig 12 ul wat for 6min; D2O
- Flow BGB 50ul twice, wait for 2 min; D2O
- Flow DNA 10 ul, wait for 12 min; D2O
- Sonicate beads 90sec
- Flow BGB 50ul twice, wait none; D2O
- Flow beads, wait for 12 min; D2O
- Flow BGB 50 ul-Thrice, wait none;
- Seal it