IGEM:Harvard/2006/vlau/Notebook/2006-8-2: Difference between revisions
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===''PEG Precipitation''=== | ===''PEG Precipitation''=== | ||
1. Goal: repeat titration of PEG precipitation conditions for 2 samples of 6hb | 1. Goal: repeat titration of PEG precipitation conditions for 2 samples of 6hb | ||
2. Protocol | 2. Protocol | ||
{| {{table}} | {| {{table}} | ||
| Line 27: | Line 28: | ||
- pipetted supernatant into separate tube | - pipetted supernatant into separate tube | ||
- pellet resuspended in 1x folding buffer volume equal to supernatant | - pellet resuspended in 1x folding buffer volume equal to supernatant | ||
- ran equivalent volumes of pellet and supernatant on 2% agarose gel (+11 mM {{mgcl2}} | - ran equivalent volumes of pellet and supernatant on 2% agarose gel (+11 mM {{mgcl2}}) | ||
3. Gel Analysis | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Lane''' | |||
| align="center" style="background:#f0f0f0;"|'''Contents''' | |||
| align="center" style="background:#f0f0f0;"|'''Loading Dye''' | |||
|- | |||
|- 1||1 kb Ladder (4 {{ul}})||- | |||
|- | |||
|- 2|| | |||
|8/1 6hb: 40 {{ul}}||3%||30 {{ul}}||20 {{ul}}||110 {{ul}}||200 {{ul}} | |||
|- | |||
|8/1 6hb: 40 {{ul}}||4%||40 {{ul}}||20 {{ul}}||100 {{ul}}||200 {{ul}} | |||
|- | |||
|8/1 6hb: 40 {{ul}}||5%||50 {{ul}}||20 {{ul}}||90 {{ul}}||200 {{ul}} | |||
|- | |||
|old 6hb: 40 {{ul}}||3%||30 {{ul}}||20 {{ul}}||110 {{ul}}||200 {{ul}} | |||
|- | |||
|old 6hb: 40 {{ul}}||4%||40 {{ul}}||20 {{ul}}||100 {{ul}}||200 {{ul}} | |||
|- | |||
|old 6hb: 40 {{ul}}||5%||50 {{ul}}||20 {{ul}}||90 {{ul}}||200 {{ul}} | |||
|- | |||
|} | |||
Revision as of 07:45, 2 August 2006
PEG Precipitation
1. Goal: repeat titration of PEG precipitation conditions for 2 samples of 6hb
2. Protocol
| Sample | % PEG | 20% PEG Volume | 5 M NaCl Volume | dH2O | Total Volume |
| 8/1 6hb: 40 μL | 3% | 30 μL | 20 μL | 110 μL | 200 μL |
| 8/1 6hb: 40 μL | 4% | 40 μL | 20 μL | 100 μL | 200 μL |
| 8/1 6hb: 40 μL | 5% | 50 μL | 20 μL | 90 μL | 200 μL |
| old 6hb: 40 μL | 3% | 30 μL | 20 μL | 110 μL | 200 μL |
| old 6hb: 40 μL | 4% | 40 μL | 20 μL | 100 μL | 200 μL |
| old 6hb: 40 μL | 5% | 50 μL | 20 μL | 90 μL | 200 μL |
- incubated on ice for 15 min.
- spun @ 16k rcf for 10 min.
- pipetted supernatant into separate tube
- pellet resuspended in 1x folding buffer volume equal to supernatant
- ran equivalent volumes of pellet and supernatant on 2% agarose gel (+11 mM MgCl2)
3. Gel Analysis
| Lane | Contents | Loading Dye | |||
| 8/1 6hb: 40 μL | 3% | 30 μL | 20 μL | 110 μL | 200 μL |
| 8/1 6hb: 40 μL | 4% | 40 μL | 20 μL | 100 μL | 200 μL |
| 8/1 6hb: 40 μL | 5% | 50 μL | 20 μL | 90 μL | 200 μL |
| old 6hb: 40 μL | 3% | 30 μL | 20 μL | 110 μL | 200 μL |
| old 6hb: 40 μL | 4% | 40 μL | 20 μL | 100 μL | 200 μL |
| old 6hb: 40 μL | 5% | 50 μL | 20 μL | 90 μL | 200 μL |
