User:Pranav Rathi/Notebook/OT/2013/01/09/DNA-Overstretching Experiments

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Introduction

This Page contains all the information regarding DNA-Overstretching experiments and results.

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More on Optical Tweezers [1]


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[2]

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: [3]

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

  1. Flow anti-dig 12 ul wat for 6min; H2O and D2O
  2. Flow BGB 50ul twice, wait for 2 min; H2O and D2O
  3. Flow DNA 10 ul, wait for 12 min; H2O and D2O
  4. Sonicate beads 90sec
  5. Flow BGB 50ul twice, wait none; H2O and D2O
  6. Flow beads, wait for 12 min; H2O and D2O
  7. Flow 1XPOP 50 ul-Twice, wait none; H2O and D2O
  8. 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: [4]

Data and other information can be seen through evernotes:[5]


<html><iframe height="300px" width=600px" src="https://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

  1. Flow anti-dig 12 ul wat for 6min; H2O and D2O
  2. Flow BGB 50ul twice, wait for 2 min; H2O and D2O
  3. Flow DNA 10 ul, wait for 12 min; H2O and D2O
  4. Sonicate beads 90sec
  5. Flow BGB 50ul twice, wait none; H2O and D2O
  6. Flow beads, wait for 12 min; H2O and D2O
  7. Flow BGB 50 ul-Twice, wait none; H2O and D2O
  8. 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:[6]

Data and other information can be seen through evernotes:[7]

<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

  1. Flow anti-dig 12 ul wat for 6min; H2O and D2O
  2. Flow BGB 50ul twice, wait for 2 min; H2O and D2O
  3. Flow DNA 10 ul, wait for 12 min; H2O and D2O
  4. Sonicate beads 90sec
  5. Flow BGB 50ul twice, wait none; H2O and D2O
  6. Flow beads, wait for 12 min; H2O and D2O
  7. Flow BGB 50 ul-Twice, wait none; H2O and D2O
  8. 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:

[8]

Overstretching-data and other information can be seen through evernotes:[9]

<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:[10]

<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

  1. Flow anti-dig 12 ul wat for 6min; H2O
  2. Flow BGB 50ul twice, wait for 2 min; H2O
  3. Flow DNA 10 ul, wait for 12 min; H2O
  4. Sonicate beads 90sec
  5. Flow BGB 50ul twice, wait none; H2O
  6. Flow beads, wait for 12 min; H2O
  7. Flow BGB 50 ul-Thrice, wait none;
  8. 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
  • Good selected segments are:(0001-4),(0002-0),(0004-7,14,15,17,18,19,20,25,26)
  • The data link at server:http:

[11]

Overstretching-data and other information can be seen through evernotes:[12]

<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:[13]

<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 D2O=15ul of bead D2O (1:50)

DNA: ds-DNA 4.4kb (110ng/ml;02/11/11)

  • D2O;

1ul of DNA from stock (1:10) + 9ul of 1XPOP D2O =10ul of DNA D2O (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.
  1. Flow anti-dig 12 ul wat for 6min; D2O
  2. Flow BGB 50ul twice, wait for 2 min; D2O
  3. Flow DNA 10 ul, wait for 12 min; D2O
  4. Sonicate beads 90sec
  5. Flow BGB 50ul twice, wait none; D2O
  6. Flow beads, wait for 12 min; D2O
  7. Flow BGB 50 ul-Thrice, wait none;
  8. 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 D2o,TCH/BH 574nm,FH 200 & 300nm.
  • Data is in file: 1301110\001 to 003.
  • Good selected 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:

[14]

Overstretching-data and other information can be seen through evernotes: [15]

<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)

  • H2O;

1ul of DNA from stock (1:10) + 9ul of 1XPOP H2O =10ul of DNA H2O (1:100)

Procedure

  • The procedure is same as experiment 5(01/10/13)

Result:

Sample is very successful tethering wise, lot of tethers, but most of them broke during the beginning of overstretching after a good FTC. Very very few tethers overstretched; 11 good tethers out of 75 attempts (15% efficiency). I selected some of the beast segments; force goes from 59 to 66pN at 1750nm LDNA at which the DNA starts overstretching but the data is good within the error range of stiffness. This data is analyzed under quick conversion, because we have not got the geometry going so the data might change.

Some tethers overstretched multiple times, i noticed every time the same tethers is overstretched the overstretching force depletes, but that is not case. I looked many many multiple overstretches and they looked very alike.

Data:
  • Data acquisition parameters: DNA4.4kb(110ng/ml; 02/11/11) ,r=265nm ,medium H2o,TCH/BH 574nm,FH 200 ,loading rate .001, Temp=75~76 degF.
  • Data is in file: 1301115\001 to 006.
  • Good selected segments are: (0000-1,10),(0001-3,10,12),(0002-2,6),(0003-0,12,13, (0004-5)
  • Repeated overstretching segments (the segments in which same tether overstretched multiple times) are:(0000-(3,4),(10,11,12)); first number shows the overstretch first time consecutive ascending number shows 2nd, 3rd, so-forth and so-on time overstretch.
  • The data link at server:http:

[16]

Overstretching-data and other information can be seen through evernotes:[17]

<html><iframe height="300px" width=600px" src="http://www.evernote.com/shard/s24/sh/b188864a-8228-4570-87d0-5234228805a1/480f4bbe5ad4223b3ff163b0b0664195"></iframe></html>

Experiment:2

Overstrething in D2O

Sample preparation

Single-chamber sample.

Bead:

  • 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)

  • D2O;

1ul of DNA from stock (1:10) + 9ul of 1XPOP D2O =10ul of DNA D2O (1:100)

Procedure

  • The procedure is same as experiment 5(01/10/13)

Result:

Sample is very successful tethering wise, lot of tethers, most of the tethers overstretched unlike the H2O sample. I got 31 good overstretches out of 84 attempts (40% efficiency); 3 times better than H2O sample. I selected some of the beast segments; force goes from 63 to 69pN at 1750nm LDNA at which the DNA starts overstretching but the data is good within the error range of stiffness. This data is analyzed under quick conversion, because we have not got the geometry going so the data might change. On average the overstretching force is reported higher in D2O than H2O.

Data:
  • Data acquisition parameters: DNA4.4kb(110ng/ml; 02/11/11) ,r=265nm ,medium D2o,TCH/BH 574nm,FH 200 ,loading rate .001, Temp=77~78 degF.
  • Data is in file: 1301115\007 to 013.
  • Good selected segments are: 0007-6, 0008-3,(0009-0,1,5,10), (0010-7,10), 0011-4, (0012-1,3,10) 0013-3.
  • Repeated overstretching segments are:

0007-(6,7),0008-(3,4),0009-(1,2,3),0011-(4,5),0012-(1,2),(3,4),(10,11),0013-(3,4)

  • The data link at server:http:

[18]

Overstretching-data and other information can be seen through evernotes:[19]

<html><iframe height="300px" width=600px" src="http://www.evernote.com/shard/s24/sh/b188864a-8228-4570-87d0-5234228805a1/480f4bbe5ad4223b3ff163b0b0664195"></iframe></html>

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