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 is in file: 1301104-0621 to 0626.
- The data link at server: 
Data and other information can be seen through evernotes: