User:Pranav Rathi/Notebook/OT/2011/01/10/DNA tethering experiments: Difference between revisions
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==Atempt .1 (== | ==Atempt .1 (jan/10/2011)== | ||
I attempt tethering again today with new beads and new anti-dig. | I attempt tethering again today with new beads and new anti-dig. | ||
===Ant-dig=== | ===Ant-dig=== | ||
Line 24: | Line 24: | ||
{{#widget:YouTube|id=A_CUeRuwxco}} | {{#widget:YouTube|id=A_CUeRuwxco}} | ||
{{#widget:YouTube|id=ehFeJ165Tis}} | {{#widget:YouTube|id=ehFeJ165Tis}} | ||
==Atempt .2 (jan/12/2011)== | |||
===components=== | |||
.5μm bead with 1.1kb dna (ant just made), duplex. | |||
* 5μl .5 μm bead + 45μl BGB =50μl (1:10). | |||
* 1.1kb dna (1:100) and (1.5:100). | |||
===Procedure[http://openwetware.org/wiki/Koch_Lab:Protocols/Microsphere-DNA_tethering/Glass,_dig,_biotin,_microsphere,_4kb_DNA]=== | |||
# Flow anti-dig 10μl wait for 5 min. | |||
# Flow BGB 50μl wait for 2 min. | |||
# Flow 1.1kb (new)10μl wait for 14 min. | |||
# Flow BGB wait none. | |||
# Flow micro-spheres one in each chamber 10μl wait for 14 min. | |||
# Flow BGB 50μl + seal it with nail polish | |||
===Results=== | |||
Both the chambers were same no difference between 1 and 1.5 to 100 concentration. Hardly any tethers. The one found, break immediately. |
Revision as of 14:10, 7 February 2011
Atempt .1 (jan/10/2011)
I attempt tethering again today with new beads and new anti-dig.
Ant-dig
Dr. Koch and I prepared new anti-dig: 1X concentration
- PBS [1]180μl + aliquots 20μl = [MIX] = Anti-dig 200μl.
PBS was used from the glass door fridge and aliquot from super cold gray fridge. I used two different beads:
- 1 μm new beads (looks good in microscope), 20μl from the batch. 5μl (batch) +4 5μl BGB = 50μl.
- .5μm (FL) expired on 01/07/2010.5μl (batch)+45μl BGB = 50μl.
Procedure[2]
- Flow anti-dig 10μl wait for 5 min.
- Flow BGB 50μl wait for 2 min.
- Flow 1.1kb (new)10μl wait for 14 min.
- Flow BGB wait none.
- Flow micro-spheres one in each chamber 10μl wait for 14 min.
- Flow BGB 50μl + seal it with nail polish
Results
1μM BEAD.[3]
{{#widget:YouTube|id=OvuG6Ju77h0}} {{#widget:YouTube|id=A_CUeRuwxco}} {{#widget:YouTube|id=ehFeJ165Tis}}
Atempt .2 (jan/12/2011)
components
.5μm bead with 1.1kb dna (ant just made), duplex.
- 5μl .5 μm bead + 45μl BGB =50μl (1:10).
- 1.1kb dna (1:100) and (1.5:100).
Procedure[4]
- Flow anti-dig 10μl wait for 5 min.
- Flow BGB 50μl wait for 2 min.
- Flow 1.1kb (new)10μl wait for 14 min.
- Flow BGB wait none.
- Flow micro-spheres one in each chamber 10μl wait for 14 min.
- Flow BGB 50μl + seal it with nail polish
Results
Both the chambers were same no difference between 1 and 1.5 to 100 concentration. Hardly any tethers. The one found, break immediately.