IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-7-27

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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-3 Neg. control for the master used in LC1 reactions
34 none none none Vent PCR-3 Neg. control for the master used in LC2 reactions
35 none none none Vent PCR-3 Neg. control for the master used in primer reactions

*Or however much template is normally used in the positive control

A black dot on the vial indicates the LC1 set (done by Jeff), a purple dot indicates LC2 (done by David). Hetmann did the primer controls.

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.

Note: we ran out of the Cat primer, so Jeff's positive control probably won't work.

Note: we made a calculation error; our master mix had 25% more water than it's supposed to. Hopefully this won't affect our results (each reaction is still the same total volume, so the concentrations of template and primer were not affected).