User:Steven J. Koch/Notebook/Kochlab/2009/07/15/dsDNA tethering
Steve Koch 21:43, 15 July 2009 (EDT): Ant setup some pRL574 dig/bio PCR yesterday and analyzed it today. His gel looked awesome, but we don't yet have photo capability. He did the same 6 reactions that Diego and I did last Spring. This time, he upped the elongation time to 4.5 minutes (I think/see Ant's notes). Reactions 1,2,3,5,6 worked. I purified these 5 reactions using QIAGEN PCR cleanup kit. Tube #6, I lost about 20 microliters of original due to stock. Otherwise, I followed the instructions, using the centrifugue in our lab at max speed. There is no color indicator in buffer PB. I eluted with 50 microliters of buffer EB. Also, I noticed that the beads, antidig, etc. rack was out on the benchtop. Probably it was out for several hours, but I'm thinking the BGB,
antidig will still be OK. (Actually, antidig was not left out.)
Linh N Le 15:03, 16 July 2009 (EDT) I apologize for leaving the beads out. I figured my sample was going to be horrendous and have to be remade. The beads were in the fridge before we used them so they were out for no more than 2-3hrs (made sample around 5 i would say and then you returned to the lab at 7)
- Steve Koch 22:58, 16 July 2009 (EDT): No worries Linh -- those things are going to happen, and it's great to have it recorded in the notebook exactly how long they were out, so we can now whether it matters in the future. (As good as Ant's dsDNA was working, it appears to me that it did not matter at all.) Also, the main reason for it happening was the extreme level of urgency I was imposing with this deadline. That kind of rushing is going to bring about those kind of mistakes, I know. Thank you for letting me know! --S
Steve Koch 22:06, 15 July 2009 (EDT): Diluting 10 microliters of purified #1 (4.4 kb) 1:10 into 90 microliters of 1x Popping buffer. Freezing the rest of #1 and the other 4 tubes as well. (Not making dilutions of those right now.) I put these into Ant's -20C box.
Following basic tethering protocol.
- antidig (from Monday), 10 microliters (sample volume), 5 minutes
- BGB Pop, 3 time 5 s.v. in succession
- DNA (1:10 dilution of today's purification of tube #1 (4.4 kb) 5 minutes (used 1 s.v.; 10 ul)
- BGB Pop, 3 times 5 s.v. in succession
- 1.5 s.v. beads (from Monday, 1:10 into BGB, sonicated, not washed) for over 10 minutes. Probably 15 minutes.
- washed 3 times 2 s.v. BGB
- sealed with nail polish @ about 9:20 pm
Way too much DNA I think! This is great news. It means I can just make another sample and reduce the concentration of DNA. I'd say there are 100's of beads per large field of view. Thus, I should decrease DNA by 10x, I think.
- Actually, was able to move over towards the tape (where DNA concentration is less) and see obvious dsDNA tethers. So, I am very confident that this DNA is great (good job Ant!)
IMPORTANT I notice while looking through eye piece that the bead focus changes substantially depending on which way the bead is being moved by piezo. This seems to have more to due with direction of movement than position, so it's likely a friction problem, not a bead angle problem. I bet it's related to the sliding problem as well.
Steve Koch 05:42, 16 July 2009 (EDT): Quick results:
- feedback close to working, but probably need a couple hours of non-tired time.
- tried ripping off a dsDNA tether, but could not. Does that place force below 65 pN?
Among other things:
- Power spectrum (stiffness) versus beam diameter with 0.5 micron beads -- it's quite possibly that a larger beam will be much stiffer, since previous info was based on 1.0 micron beads
- Lower concentration (1:10 of the 1:10) of Ant's dsDNA
- higher concentration of the unzipping constructs
- Fix force clamp / and or re-write FTC so it can work w/o clamping