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

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Contents

Introduction

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

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.



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

  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: [3]

Data and other information can be seen through evernotes:

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:[4]

Data and other information can be seen through evernotes:


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:

[5]

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

Find tether center steps can be seen through this link:

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
  • The data link at server:http:

[6]

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

Find tether center steps can be seen through this link:


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 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:

[7]

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


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.

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