IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-7-27: Difference between revisions
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| align="center" style="background:#f0f0f0;"|'''Primer''' | | align="center" style="background:#f0f0f0;"|'''Primer''' | ||
| align="center" style="background:#f0f0f0;"|'''Polymerase''' | | align="center" style="background:#f0f0f0;"|'''Polymerase''' | ||
| align="center" style="background:#f0f0f0;"|'''PCR machine''' | |||
| align="center" style="background:#f0f0f0;"|'''Notes''' | |||
|- | |- | ||
| 1||LC1||10 ng||3-10 (crossF and eco1F)||Vent | | 1||LC1||10 ng||3-10 (crossF and eco1F)||Vent||PCR-5|| | ||
|- | |- | ||
| 2||LC1||10 ng||9-8 (eco1R and pst2F)||Vent | | 2||LC1||10 ng||9-8 (eco1R and pst2F)||Vent||PCR-5|| | ||
|- | |- | ||
| 3||LC1||10 ng||6-7 (pst1F and pst2R)||Vent | | 3||LC1||10 ng||6-7 (pst1F and pst2R)||Vent||PCR-3|| | ||
|- | |- | ||
| 4||LC1||10 ng||5-4 (pst1R and crossR)||Vent | | 4||LC1||10 ng||5-4 (pst1R and crossR)||Vent||PCR-5|| | ||
|- | |- | ||
| 5||LC1||5 ng||3-10||Vent | | 5||LC1||5 ng||3-10||Vent||PCR-5|| | ||
|- | |- | ||
| 6||LC1||5 ng||9-8||Vent | | 6||LC1||5 ng||9-8||Vent||PCR-5|| | ||
|- | |- | ||
| 7||LC1||5 ng||6-7||Vent | | 7||LC1||5 ng||6-7||Vent||PCR-3|| | ||
|- | |- | ||
| 8||LC1||5 ng||5-4||Vent | | 8||LC1||5 ng||5-4||Vent||PCR-5|| | ||
|- | |- | ||
| 9||LC1||1 ng||3-10||Vent | | 9||LC1||1 ng||3-10||Vent||PCR-5|| | ||
|- | |- | ||
| 10||LC1||1 ng||9-8||Vent | | 10||LC1||1 ng||9-8||Vent||PCR-5|| | ||
|- | |- | ||
| 11||LC1||1 ng||6-7||Vent | | 11||LC1||1 ng||6-7||Vent||PCR-3|| | ||
|- | |- | ||
| 12||LC1||1 ng||5-4||Vent | | 12||LC1||1 ng||5-4||Vent||PCR-5|| | ||
|- | |- | ||
| 13||LC2||10 ng||3-10||Vent | | 13||LC2||10 ng||3-10||Vent||PCR-5|| | ||
|- | |- | ||
| 14||LC2||10 ng||9-8||Vent | | 14||LC2||10 ng||9-8||Vent||PCR-5|| | ||
|- | |- | ||
| 15||LC2||10 ng||6-7||Vent | | 15||LC2||10 ng||6-7||Vent||PCR-3|| | ||
|- | |- | ||
| 16||LC2||10 ng||5-4||Vent | | 16||LC2||10 ng||5-4||Vent||PCR-5|| | ||
|- | |- | ||
| 17||LC2||5 ng||3-10||Vent | | 17||LC2||5 ng||3-10||Vent||PCR-5|| | ||
|- | |- | ||
| 18||LC2||5 ng||9-8||Vent | | 18||LC2||5 ng||9-8||Vent||PCR-5|| | ||
|- | |- | ||
| 19||LC2||5 ng||6-7||Vent | | 19||LC2||5 ng||6-7||Vent||PCR-3|| | ||
|- | |- | ||
| 20||LC2||5 ng||5-4||Vent | | 20||LC2||5 ng||5-4||Vent||PCR-5|| | ||
|- | |- | ||
| 21||LC2||1 ng||3-10||Vent | | 21||LC2||1 ng||3-10||Vent||PCR-5|| | ||
|- | |- | ||
| 22||LC2||1 ng||9-8||Vent | | 22||LC2||1 ng||9-8||Vent||PCR-5|| | ||
|- | |- | ||
| 23||LC2||1 ng||6-7||Vent | | 23||LC2||1 ng||6-7||Vent||PCR-3|| | ||
|- | |- | ||
| 24||LC2||1 ng||5-4||Vent | | 24||LC2||1 ng||5-4||Vent||PCR-5|| | ||
|- | |- | ||
| 25||none||none||3-10 (crossF and eco1F)||Vent | | 25||none||none||3-10 (crossF and eco1F)||Vent||PCR-5||Neg. control for primers | ||
|- | |- | ||
| 26||none||none||9-8 (eco1R and pst2F)||Vent | | 26||none||none||9-8 (eco1R and pst2F)||Vent||PCR-5||Neg. control for primers | ||
|- | |- | ||
| 27||none||none||6-7 (pst1F and pst2R)||Vent | | 27||none||none||6-7 (pst1F and pst2R)||Vent||PCR-3||Neg. control for primers | ||
|- | |- | ||
| 29||none||none||5-4 (pst1R and crossR)||Vent | | 29||none||none||5-4 (pst1R and crossR)||Vent||PCR-5||Neg. control for primers | ||
|- | |- | ||
| 30||Cat||10 ng*||Cat primers||Vent | | 30||Cat||10 ng*||Cat primers||Vent||PCR-3||Pos. control for the master used in LC1 reactions | ||
|- | |||
| 31||Cat||10 ng*||Cat primers||Vent||PCR-5||Pos. control for the master used in LC2 reactions | |||
|- | |||
| 32||Cat||10 ng*||Cat primers||Vent||PCR-5||Pos. control for the master used in primer reactions | |||
|- | |||
| 33||none||none||none||Vent||PCR-5||Neg. control for the master used in LC1 reactions | |||
|- | |||
| 34||none||none||none||Vent||PCR-5||Neg. control for the master used in LC2 reactions | |||
|- | |||
| 35||none||none||none||Vent||PCR-5||Neg. control for the master used in primer reactions | |||
|- | |- | ||
| | | | ||
|} | |} | ||
<nowiki> | <nowiki> | ||
*Or however much template is normally used in the positive control | *Or however much template is normally used in the positive control | ||
Revision as of 18:13, 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).
We've chosen to use LC1 3kb #1 and LC2 3kb #1 as our samples.
| Reaction # | Template | T, amount | Primer | Polymerase | PCR machine | Notes |
| 1 | LC1 | 10 ng | 3-10 (crossF and eco1F) | Vent | PCR-5 | |
| 2 | LC1 | 10 ng | 9-8 (eco1R and pst2F) | Vent | PCR-5 | |
| 3 | LC1 | 10 ng | 6-7 (pst1F and pst2R) | Vent | PCR-3 | |
| 4 | LC1 | 10 ng | 5-4 (pst1R and crossR) | Vent | PCR-5 | |
| 5 | LC1 | 5 ng | 3-10 | Vent | PCR-5 | |
| 6 | LC1 | 5 ng | 9-8 | Vent | PCR-5 | |
| 7 | LC1 | 5 ng | 6-7 | Vent | PCR-3 | |
| 8 | LC1 | 5 ng | 5-4 | Vent | PCR-5 | |
| 9 | LC1 | 1 ng | 3-10 | Vent | PCR-5 | |
| 10 | LC1 | 1 ng | 9-8 | Vent | PCR-5 | |
| 11 | LC1 | 1 ng | 6-7 | Vent | PCR-3 | |
| 12 | LC1 | 1 ng | 5-4 | Vent | PCR-5 | |
| 13 | LC2 | 10 ng | 3-10 | Vent | PCR-5 | |
| 14 | LC2 | 10 ng | 9-8 | Vent | PCR-5 | |
| 15 | LC2 | 10 ng | 6-7 | Vent | PCR-3 | |
| 16 | LC2 | 10 ng | 5-4 | Vent | PCR-5 | |
| 17 | LC2 | 5 ng | 3-10 | Vent | PCR-5 | |
| 18 | LC2 | 5 ng | 9-8 | Vent | PCR-5 | |
| 19 | LC2 | 5 ng | 6-7 | Vent | PCR-3 | |
| 20 | LC2 | 5 ng | 5-4 | Vent | PCR-5 | |
| 21 | LC2 | 1 ng | 3-10 | Vent | PCR-5 | |
| 22 | LC2 | 1 ng | 9-8 | Vent | PCR-5 | |
| 23 | LC2 | 1 ng | 6-7 | Vent | PCR-3 | |
| 24 | LC2 | 1 ng | 5-4 | Vent | PCR-5 | |
| 25 | none | none | 3-10 (crossF and eco1F) | Vent | PCR-5 | Neg. control for primers |
| 26 | none | none | 9-8 (eco1R and pst2F) | Vent | PCR-5 | Neg. control for primers |
| 27 | none | none | 6-7 (pst1F and pst2R) | Vent | PCR-3 | Neg. control for primers |
| 29 | none | none | 5-4 (pst1R and crossR) | Vent | PCR-5 | Neg. control for primers |
| 30 | Cat | 10 ng* | Cat primers | Vent | PCR-3 | Pos. control for the master used in LC1 reactions |
| 31 | Cat | 10 ng* | Cat primers | Vent | PCR-5 | Pos. control for the master used in LC2 reactions |
| 32 | Cat | 10 ng* | Cat primers | Vent | PCR-5 | Pos. control for the master used in primer reactions |
| 33 | none | none | none | Vent | PCR-5 | Neg. control for the master used in LC1 reactions |
| 34 | none | none | none | Vent | PCR-5 | Neg. control for the master used in LC2 reactions |
| 35 | none | none | none | Vent | PCR-5 | Neg. control for the master used in primer reactions |
*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.
