User:Pranav Rathi/Notebook/OT/2013/01/09/DNA-Overstretching Experiments
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.
Experiment 1 (01/04/2013)
Prepare some new buffers and attempt DNA-tethering in H2O and D2O with 265nm beads (bead radius).
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
50mg of BGB + 10ml of H2O 1X POP=5mg/ml BGB 1XPOP H2O 10ml
50mg of BGB + 10ml of D2O 1X POP=5mg/ml BGB 1XPOP D2O 10ml
Same for both H2O and D2O since PBS is in H2O only.
20ul of aliquots + 180ul of PBS H2O=200ul of antidig H2O
Dual-chamber sample. For more information go to sample preparation for DNA overstretching & unzipping experiments: 
1.5ul of 265nm bead (1:5) from stock+13.5ul of BGB H2O=15ul of bead H2O (1:5o)
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)
1ul of DNA from stock (1:10) + 9ul of 1XPOP H2O =10ul of DNA H2O (1:100)
1ul of DNA from stock (1:10) + 9ul of 1XPOP D2O =10ul of DNA D2O (1:100)
- 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
- 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.
With 520nm big beads flow 1XPOP in the last step with 265nm small beads flow BGB in the last step.
Data and other information can be seen through evernotes: