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:

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