IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-11

Thoughts/ramblings/goals/questions/general frustrations

The results of yesterday's experiment show that Microcon filtration gives low yields and the PEG precipitation (at least at 10%) damages nanostructures regardless of folding conditions.

Questions

• Are Microcon yields unacceptably low, or are they acceptable? (Can we use the NanoDrop to quantify our yield?)
  • Gels are much better for quantifying yield - you can try both and see how they compare.
• Low concentrations of PEG should precipitate large nanostructures. Will some smaller concentration of PEG not harm nanostructures formed under some folding conditions?
  • Our August 2 experiment showed that even low concentrations of PEG damage nanostructures folded under "standard" conditions (10x oligos, 10 mM MgCl₂).

p7308 quantitation

• Speedvac 060522 p7308 sample down to 50% volume. This should remove any ethanol, and give you a slightly more manageable volume.
• Pour 2% agraose, 11 mM MgCl₂ gel
• For gel loading make 1:2 dilution (add 20 μL of p7308 to 20 μL dH₂O). Original estimate for 060522 prep was 42 nM, so hopefully it should correspond pretty well to 44 nM sample.
• When imaging gel, use spot density tool to measure intensity of each band
  • Use saturation indicator to take a picture just below the point where any bands start saturating on the image
  • Draw a rectangle that fits around the largest band on the gel
  • Copy that rectangle and position it directly above the first band. This will be used to measure background
  • Repeat this for every band on the gel (one box for the band, one box for background)
  • Record this data along with gel picture on the wiki
• To determine p7308 concentration, use background-subtracted value for each volume. Scale each control concentration (44 nM) according to the ratio of background-subtracted intensity for the band that looks closest in intensity
  • For example, if the band in lane 1 (3 μL, 44 nM) had an intensity of 1000, and the band in lane 5 (3 μL, ?? nM) had an intensity of 900, then we would record 900/1000 * 44 = 39.6 nM as the estimated concentration for that lane. Repeating for each lane should give you 3 data points, which you can average (throwing out any obvious outliers).

File:IGEM060811-p7308a.jpg

2% agarose gel, 0.5 mg/mL EtBr
0.5x TBE, 11 mM MgCl₂

Lane	Contents	Loading Buffer
0	1kb DNA ladder (5 μL)
1	p7308 060323, 44 nM (3 μL)	AGLB (2 μL) + dH2O (9 μL)
2	p7308 060323, 44 nM (6 μL)	AGLB (2 μL) + dH2O (6 μL)
3	p7308 060323, 44 nM (9 μL)	AGLB (2 μL) + dH2O (3 μL)
4	p7308 060522 (1 μL)	AGLB (2 μL) + dH2O (11 μL)
5	p7308 060522 (3 μL)	AGLB (2 μL) + dH2O (9 μL)
6	p7308 060522 (6 μL)	AGLB (2 μL) + dH2O (6 μL)
7	p7308 060522 (9 μL)	AGLB (2 μL) + dH2O (3 μL)
8	p7308 060522 (12 μL)	AGLB (2 μL)