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Team:Stanford/Notebook/Lab Work/Week 7
From 2010.igem.org
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troubleshooting PCR stitching reactions (prior to ligations)- recommendations from Christina: | troubleshooting PCR stitching reactions (prior to ligations)- recommendations from Christina: | ||
| - | *each "step:" try | + | *each "step:" try RSID2, sRNA1C; use best result to continue to next "step" |
#different starting DNA concentrations: 0.25X, 0.5X, 1X, 2X | #different starting DNA concentrations: 0.25X, 0.5X, 1X, 2X | ||
#different annealing temperatures: 50oC, 55oC, 60oC | #different annealing temperatures: 50oC, 55oC, 60oC | ||
| Line 65: | Line 65: | ||
Step 1: try different DNA concentrations | Step 1: try different DNA concentrations | ||
| + | *Francisco set up tubes and added PCR SuperMix (10 uL reactions) | ||
| + | *I added primers to 1 series of 4, he did the other | ||
| + | *run on 2% gel at 90V for 30 minutes | ||
== 8/12 Thursday == | == 8/12 Thursday == | ||
Revision as of 22:40, 11 August 2010
| Home | Project | Applications | Modeling | Parts | Team | Notebook |
Spring: Brainstorming | Spring Meetings
Summer: Week 1 | Week 2 | Week 3 | Week 4 | Week 5 | Week 6 | Week 7 | Week 8 | Week 9 | Week 10 | Summaries
|
8/9 Monday
Laura's Notebook
- helped Alex NanoDrop some of his samples (concentrations also written on tops of tubes)
| 260/280 | 260/230 | ng/uL | |
| sGFP mDNA | 1.71 | 1.01 | 320.3 |
| sGFP mDNA s | 1.91 | 1.41 | 220.7 |
| F2620 | 1.10 | 0.87 | 45.0 |
| F2620 s | 1.52 | 0.88 | 53.1 |
| I5 | 1.90 | 1.06 | 165.8 |
| I5s | 1.98 | 1.22 | 113.9 |
| I4 | 1.73 | 0.95 | 384.2 |
| I0500 | 1.86 | 0.89 | 67.7 |
| C1 | 1.77 | 0.80 | 83.0 |
| C2 | 1.83 | 0.83 | 86.6 |
| C3 | 1.95 | 1.09 | 75.7 |
8/10 Tuesday
Laura's Notebook
set up colony PCR for single colony from pLUX-sRNA1C ligation/transformation:
- 9 uL supermix, 0.5 uL of each primer (VF, VR)
- cycling program as before: COLPCR72
3pm meeting with Drew, Rayka about device names, parameters
8/11 Wednesday
Laura's Notebook
miniprepped pBAD, pLUX
- used Promega kit
- combined 2 liquid cultures for each into 1 tube (when pelleting cells)
- eluted in 50 uL H2O (from kit)
set up colony PCR (same as yesterday; tube dried up in machine)
troubleshooting PCR stitching reactions (prior to ligations)- recommendations from Christina:
- each "step:" try RSID2, sRNA1C; use best result to continue to next "step"
- different starting DNA concentrations: 0.25X, 0.5X, 1X, 2X
- different annealing temperatures: 50oC, 55oC, 60oC
- cycle numbers: 5, 10, 15, 20
Step 1: try different DNA concentrations
- Francisco set up tubes and added PCR SuperMix (10 uL reactions)
- I added primers to 1 series of 4, he did the other
- run on 2% gel at 90V for 30 minutes
