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

Introduction

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

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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}}

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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

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)