IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-9: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
No edit summary |
|||
Line 16: | Line 16: | ||
==Microcon purification of yesterday's folding reactions== | ==Microcon purification of yesterday's folding reactions== | ||
*speedvac'd c3.2F, G, H, I, Eb, Fb flowthrough for 50 min. (vol. def much higher than 40 {{ul}}) | |||
*ran 2% agarose + {{mgcl2}} gel | |||
[[Image:2006-08-09 microcon1.jpg|thumb|2% agarose gel, 0.5 mg/mL EtBr<br>0.5x TBE, 11 mM {{mgcl2}}]] | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Lane''' | |||
| align="center" style="background:#f0f0f0;"|'''Contents''' | |||
| align="center" style="background:#f0f0f0;"|'''Loading Buffer''' | |||
|- | |||
| 0||1kb ladder (10 {{ul}})||- | |||
|- | |||
| 1||naked p7308 (7.5 {{ul}})||5 {{ul}} | |||
|- | |||
| 2||c3.2F (7.5 {{ul}})||5 {{ul}} | |||
|- | |||
| 3||c3.2F flowthrough (35 {{ul}})||5 {{ul}} | |||
|- | |||
| 4||c3.2G (7.5 {{ul}})||5 {{ul}} | |||
|- | |||
| 5||c3.2G flowthrough (35 {{ul}})||5 {{ul}} | |||
|- | |||
| 6||c3.2H (7.5 {{ul}})||5 {{ul}} | |||
|- | |||
| 7||c3.2H flowthrough (35 {{ul}})||5 {{ul}} | |||
|- | |||
| 8||c3.2I (7.5 {{ul}})||5 {{ul}} | |||
|- | |||
| 9||c3.2I flowthrough (35 {{ul}})||5 {{ul}} | |||
|- | |||
| 10||c5.0Eb (7.5 {{ul}})||5 {{ul}} | |||
|- | |||
| 11||c5.0Eb flowthrough (35 {{ul}})||5 {{ul}} | |||
|- | |||
| 12||c5.0Fb (7.5 {{ul}})||5 {{ul}} | |||
|- | |||
| 13||c5.0Fb flowthrough (35 {{ul}})||5 {{ul}} | |||
|} | |||
*3 flowthroughs is probably not enough - notice pretty clear oligo smears: should probably do 5/6 | |||
*don't know if oligo-ligand and latches were added to rxns before microcon: repeat folding of c3.2F, G, H, I and retest |
Revision as of 10:55, 9 August 2006
Folding reaction
Fold 200 μL each of 3.2.Eo, .Fo, .Go, .Ho
Each reaction:
- 45 μL p7308 scaffold (44 nM)
- 80 μL oligo stock (250 nM)
- 20 μL 10x folding buffer (500 mM HEPES pH 7.5, 500 mM NaCl, 100 mM MgCl2)
- 55 μL dH2O
Folding conditions:
- high-volume protocol: start at 80[[:Category:{{{1}}}|{{{1}}}]], decrement 0.5[[:Category:{{{1}}}|{{{1}}}]] every 1 min. for 120 cycles.
More Microcon trials
Microcon purification of yesterday's folding reactions
- speedvac'd c3.2F, G, H, I, Eb, Fb flowthrough for 50 min. (vol. def much higher than 40 μL)
- ran 2% agarose + MgCl2 gel
Lane | Contents | Loading Buffer |
0 | 1kb ladder (10 μL) | - |
1 | naked p7308 (7.5 μL) | 5 μL |
2 | c3.2F (7.5 μL) | 5 μL |
3 | c3.2F flowthrough (35 μL) | 5 μL |
4 | c3.2G (7.5 μL) | 5 μL |
5 | c3.2G flowthrough (35 μL) | 5 μL |
6 | c3.2H (7.5 μL) | 5 μL |
7 | c3.2H flowthrough (35 μL) | 5 μL |
8 | c3.2I (7.5 μL) | 5 μL |
9 | c3.2I flowthrough (35 μL) | 5 μL |
10 | c5.0Eb (7.5 μL) | 5 μL |
11 | c5.0Eb flowthrough (35 μL) | 5 μL |
12 | c5.0Fb (7.5 μL) | 5 μL |
13 | c5.0Fb flowthrough (35 μL) | 5 μL |
- 3 flowthroughs is probably not enough - notice pretty clear oligo smears: should probably do 5/6
- don't know if oligo-ligand and latches were added to rxns before microcon: repeat folding of c3.2F, G, H, I and retest