IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-7-27: Difference between revisions
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==Miniprep of LC1PUR1 and LC2PUR1 transformants== | |||
We miniprepped the transformants we [[IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-7-26#Inoculation_of_LC1PUR1_and_LC2PUR1_transformants|inoculated yesterday]]. There are 8 total. We've recorded their concentrations below (we dropped the "PUR1" from the ends of their names). | |||
All DNA was eluted in 50 µL H2O. | |||
==PCR mutagenesis #6== | ==PCR mutagenesis #6== | ||
The main purpose of this PCR is to determine the optimal concentration of template. The smearing in our previous PCRs suggests that we've been using too much template (or our primers are contaminated). | The main purpose of this PCR is to determine the optimal concentration of template. The smearing in our previous PCRs suggests that we've been using too much template (or our primers are contaminated). | ||
Revision as of 17:00, 27 July 2006
Miniprep of LC1PUR1 and LC2PUR1 transformants
We miniprepped the transformants we inoculated yesterday. There are 8 total. We've recorded their concentrations below (we dropped the "PUR1" from the ends of their names).
All DNA was eluted in 50 µL H2O.
PCR mutagenesis #6
The main purpose of this PCR is to determine the optimal concentration of template. The smearing in our previous PCRs suggests that we've been using too much template (or our primers are contaminated).
| Reaction # | Template | T, amount | Primer | Polymerase |
| 1 | LC1 | 10 ng | 3-10 (crossF and eco1F) | Vent |
| 2 | LC1 | 10 ng | 9-8 (eco1R and pst2F) | Vent |
| 3 | LC1 | 10 ng | 6-7 (pst1F and pst2R) | Vent |
| 4 | LC1 | 10 ng | 5-4 (pst1R and crossR) | Vent |
| 5 | LC1 | 5 ng | 3-10 | Vent |
| 6 | LC1 | 5 ng | 9-8 | Vent |
| 7 | LC1 | 5 ng | 6-7 | Vent |
| 8 | LC1 | 5 ng | 5-4 | Vent |
| 9 | LC1 | 1 ng | 3-10 | Vent |
| 10 | LC1 | 1 ng | 9-8 | Vent |
| 11 | LC1 | 1 ng | 6-7 | Vent |
| 12 | LC1 | 1 ng | 5-4 | Vent |
| 13 | LC2 | 10 ng | 3-10 | Vent |
| 14 | LC2 | 10 ng | 9-8 | Vent |
| 15 | LC2 | 10 ng | 6-7 | Vent |
| 16 | LC2 | 10 ng | 5-4 | Vent |
| 17 | LC2 | 5 ng | 3-10 | Vent |
| 18 | LC2 | 5 ng | 9-8 | Vent |
| 19 | LC2 | 5 ng | 6-7 | Vent |
| 20 | LC2 | 5 ng | 5-4 | Vent |
| 21 | LC2 | 1 ng | 3-10 | Vent |
| 22 | LC2 | 1 ng | 9-8 | Vent |
| 23 | LC2 | 1 ng | 6-7 | Vent |
| 24 | LC2 | 1 ng | 5-4 | Vent |
| 25 | none | none | 3-10 (crossF and eco1F) | Vent |
| 26 | none | none | 9-8 (eco1R and pst2F) | Vent |
| 27 | none | none | 6-7 (pst1F and pst2R) | Vent |
| 29 | none | none | 5-4 (pst1R and crossR) | Vent |
| 30 | Cat | 10 ng* | Cat primers | Vent |
*Or however much template is normally used in the positive control
Note: I didn't include negative controls with template but no primer or Vent, since I think those controls should wait until we discover the appropriate concentration of template to use.
