IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-7-25: Difference between revisions
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==Oligos Order Form== | |||
[[c5.0 Partial Oligo Order Form]] | |||
==Purification== | |||
====PEG precipitation==== | |||
* Used samples of 3.2.H and 3.2.I [[IGEM:Harvard/2006/DNA_nanostructures/Notebook/2006-7-24#Folding_reaction|folded yesterday]] | |||
* 20 {{ul}} of each, 190 mL water, 50 mL 20% PEG / 2.5 M NaCl in respective 500 {{ul}} PCR tubes | |||
[[Image:20060725_peg.jpg|thumb|]] | |||
* gel analysis ({{aggel}}): | |||
** lanes 3 and 6 (reconstituted precipitate of H and I, respectively) appear to be free of oligos (compare to lanes 2 and 5, which are raw folding reaction), but yields are still very low, as evidenced by amount of nanostructures in supernatant (lanes 4 and 7) | |||
====Gel purification==== | |||
* 20 mL each of 3.2.D and 3.2.E run on a {{aggel}} gel | |||
* nanostructure bands isolated and purified using Qiagen gel purification kit (good for fragments up to 10 kb according to manufacturer) | |||
* will run on agarose gel tomorrow to determine purity | |||
==Silver staining experiments== | ==Silver staining experiments== | ||
* goal: to determine the minimum concentration of streptavidin that can be visualized with silver stain, and to note the times at which different concentrations become visible | * goal: to determine the minimum concentration of streptavidin that can be visualized with silver stain, and to note the times at which different concentrations become visible | ||
* methods: 12% PA gel with lanes listed below, run for 20 min. at 130 V, then stained with [http://bio-rad.com/B2B/BioRad/product/br_category.jsp?BV_SessionID=@@@@0044032730.1153852982@@@@&BV_EngineID=cccdaddiglfhhimcfngcfkmdhkkdfll.0&categoryPath=%2fCatalogs%2fLife+Science+Education%2fEquipment+and+Supplies%2fProteins+and+Proteomic+Studies%3a+Equipment+and+Reagents%2fProtein+Stains%2fSilver+Stain+Plus+Kit&catLevel=6&divName=Corporate&loggedIn=false&lang=English&country=HQ&catOID=-19568&isPA=false&serviceLevel=Lit+Request Bio-Rad Silver Stain Plus Kit] and [http://bio-rad.com/LifeScience/pdf/Bulletin_9056.pdf protocol]. | * methods: 12% PA gel with lanes listed below, run for 20 min. at 130 V, then stained with [http://bio-rad.com/B2B/BioRad/product/br_category.jsp?BV_SessionID=@@@@0044032730.1153852982@@@@&BV_EngineID=cccdaddiglfhhimcfngcfkmdhkkdfll.0&categoryPath=%2fCatalogs%2fLife+Science+Education%2fEquipment+and+Supplies%2fProteins+and+Proteomic+Studies%3a+Equipment+and+Reagents%2fProtein+Stains%2fSilver+Stain+Plus+Kit&catLevel=6&divName=Corporate&loggedIn=false&lang=English&country=HQ&catOID=-19568&isPA=false&serviceLevel=Lit+Request Bio-Rad Silver Stain Plus Kit] and [http://bio-rad.com/LifeScience/pdf/Bulletin_9056.pdf protocol]. | ||
* | ** 35 min. in fixative solution | ||
** | ** 2 x 10 min. wash in water | ||
** XX min. in staining solution | |||
** 15 min. in stopping solution | |||
[[Image:20060725_silver_strep.jpg|thumb|The streptavidin band appears at half the motility of the dye band (which was bleached to white after silver staining).]] | |||
{| {{table}} | |||
| align="center" rowspan=2 style="background:#f0f0f0;"|'''lane''' | |||
| align="center" colspan=2 style="background:#f0f0f0;"|'''streptavidin''' | |||
| align="center" rowspan=2 style="background:#f0f0f0;"|'''water''' ({{ul}}) | |||
| align="center" rowspan=2 style="background:#f0f0f0;"|'''10x Tris-gly loading dye''' ({{ul}}) | |||
|- | |||
| align="center"|({{ul}})|| align="center"|(fmols) | |||
|- | |||
| 1||10 {{ul}} 4 {{um}}||40,000||0||2 | |||
|- | |||
| 2||10 {{ul}} 2 {{um}}||20,000||0||2 | |||
|- | |||
| 3||5 {{ul}} 2 {{um}}||10,000||5||2 | |||
|- | |||
| 4||5 {{ul}} 1 {{um}}||5,000||5||2 | |||
|- | |||
| 5||5 {{ul}} 0.5 {{um}}||2,500||5||2 | |||
|- | |||
| 6||2 {{ul}} 0.5 {{um}}||1,000||8||2 | |||
|- | |||
| 7||1 {{ul}} 0.5 {{um}}||500||9||2 | |||
|- | |||
| 8||2.5 {{ul}} 0.1 {{um}}||250||7.5||2 | |||
|- | |||
| 9||1 {{ul}} 0.1 {{um}}||100||9||2 | |||
|- | |||
| 10||1 {{ul}} 0.05 {{um}}||50||9||2 | |||
|} | |||
* results: | |||
** first three lanes (40k, 20k, 10k pmol) stain reliably | |||
** fourth and fifth lane lane (5k, 2.5k pmol) barely stains | |||
** the rest of the lanes (1.25k pmol and less) do not stain more than the background | |||
** this is disappointing (we can image 1 pmol of streptavidin with Coomassie blue) |
Latest revision as of 06:31, 27 July 2006
Oligos Order Form
Purification
PEG precipitation
- Used samples of 3.2.H and 3.2.I folded yesterday
- 20 μL of each, 190 mL water, 50 mL 20% PEG / 2.5 M NaCl in respective 500 μL PCR tubes
- gel analysis (2% agarose):
- lanes 3 and 6 (reconstituted precipitate of H and I, respectively) appear to be free of oligos (compare to lanes 2 and 5, which are raw folding reaction), but yields are still very low, as evidenced by amount of nanostructures in supernatant (lanes 4 and 7)
Gel purification
- 20 mL each of 3.2.D and 3.2.E run on a 2% agarose gel
- nanostructure bands isolated and purified using Qiagen gel purification kit (good for fragments up to 10 kb according to manufacturer)
- will run on agarose gel tomorrow to determine purity
Silver staining experiments
- goal: to determine the minimum concentration of streptavidin that can be visualized with silver stain, and to note the times at which different concentrations become visible
- methods: 12% PA gel with lanes listed below, run for 20 min. at 130 V, then stained with Bio-Rad Silver Stain Plus Kit and protocol.
- 35 min. in fixative solution
- 2 x 10 min. wash in water
- XX min. in staining solution
- 15 min. in stopping solution
lane | streptavidin | water (μL) | 10x Tris-gly loading dye (μL) | |
(μL) | (fmols) | |||
1 | 10 μL 4 μM | 40,000 | 0 | 2 |
2 | 10 μL 2 μM | 20,000 | 0 | 2 |
3 | 5 μL 2 μM | 10,000 | 5 | 2 |
4 | 5 μL 1 μM | 5,000 | 5 | 2 |
5 | 5 μL 0.5 μM | 2,500 | 5 | 2 |
6 | 2 μL 0.5 μM | 1,000 | 8 | 2 |
7 | 1 μL 0.5 μM | 500 | 9 | 2 |
8 | 2.5 μL 0.1 μM | 250 | 7.5 | 2 |
9 | 1 μL 0.1 μM | 100 | 9 | 2 |
10 | 1 μL 0.05 μM | 50 | 9 | 2 |
- results:
- first three lanes (40k, 20k, 10k pmol) stain reliably
- fourth and fifth lane lane (5k, 2.5k pmol) barely stains
- the rest of the lanes (1.25k pmol and less) do not stain more than the background
- this is disappointing (we can image 1 pmol of streptavidin with Coomassie blue)