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 ┬ÁL 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
1LC110 ng3-10 (crossF and eco1F)VentPCR-5
2LC110 ng9-8 (eco1R and pst2F)VentPCR-5
3LC110 ng6-7 (pst1F and pst2R)VentPCR-3
4LC110 ng5-4 (pst1R and crossR)VentPCR-5
5LC15 ng3-10VentPCR-5
6LC15 ng9-8VentPCR-5
7LC15 ng6-7VentPCR-3
8LC15 ng5-4VentPCR-5
9LC11 ng3-10VentPCR-5
10LC11 ng9-8VentPCR-5
11LC11 ng6-7VentPCR-3
12LC11 ng5-4VentPCR-5
13LC210 ng3-10VentPCR-5
14LC210 ng9-8VentPCR-5
15LC210 ng6-7VentPCR-3
16LC210 ng5-4VentPCR-5
17LC25 ng3-10VentPCR-5
18LC25 ng9-8VentPCR-5
19LC25 ng6-7VentPCR-3
20LC25 ng5-4VentPCR-5
21LC21 ng3-10VentPCR-5
22LC21 ng9-8VentPCR-5
23LC21 ng6-7VentPCR-3
24LC21 ng5-4VentPCR-5
25nonenone3-10 (crossF and eco1F)VentPCR-5Neg. control for primers
26nonenone9-8 (eco1R and pst2F)VentPCR-5Neg. control for primers
27nonenone6-7 (pst1F and pst2R)VentPCR-3Neg. control for primers
29nonenone5-4 (pst1R and crossR)VentPCR-5Neg. control for primers
30Cat10 ng*Cat primersVentPCR-3Pos. control for the master used in LC1 reactions
31Cat10 ng*Cat primersVentPCR-5Pos. control for the master used in LC2 reactions
32Cat10 ng*Cat primersVentPCR-5Pos. control for the master used in primer reactions
33nonenonenoneVentPCR-3Neg. control for the master used in LC1 reactions
34nonenonenoneVentPCR-3Neg. control for the master used in LC2 reactions
35nonenonenoneVentPCR-3Neg. 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).
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