IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-23
<html><style type='text/css'> .tabs {
font-size:80%;
font-weight:none;
width: 100%;
color: #FFFFFF;
background:#FFFFFF url("/images/5/54/DarkgreenTab-bg.gif") repeat-x bottom;
}
.tabs li {
background:url("/images/3/36/DarkgeenTab-left.gif") no-repeat left top;
}
.tabs a,.tabs strong {
background:url("/images/d/d3/DarkgreenTab-right.gif") no-repeat right top;
color:#FFFFFF;
padding: 3px 10px 3px 4px;
}
.tabs strong{
color:#CCFF00;
background-image:url("/images/b/b1/DarkgreenTab-right_on.gif");
}
.tabs a:hover{
color:#66FF00;
}
</style></html>
Ligation test
With new J04500 /SP and new KaiC /XP, attempting ligation again; this time following up on the advice Roche gave me about using highly concentrated DNA and 2uL of DNA dilution buffer.
The NEB Kit says not to go above 10ng/uL rxn, or otherwise linear DNA forms... but for KaiC I'm going to try it because lower concentrations have consistently not worked - I'll stick to 300ng total sample, or 150% overestimation.
- J04500 /SP + KaiC /XP concentrated from 8/21.
- BB vector: (110.6ng @ 110.6 ng/uL) = 1 uL
- BB insert: 3*(~2100/3400)*110.6 = 204.9 ng @ (60 ng/uL) = 3.42 uL
- 1x DNA Dilution buffer: 2uL
- ddH20: 3.58uL
- J04500 /SP + KaiA /SP from 8/22 (Labeled C2)
- BB vector: 110.6ng @ (110.6 ng/uL) = 1 uL
- BB insert: 3*(~2100/3400)*110.6 = 204.9 ng @ (24.2ng/uL) = 8.47 uL Scaled to 7uL
- 1x DNA Dilution buffer: 2uL
- J04500 /SP + KaiA /SP from 8/22 (Labeled C1), concentrated down to ~5uL from 30uL
- BB vector: 330ng @ (110.6 ng/uL) = 3 uL
- BB insert: 8uL of C1 DNA, around 60ng/uL = 480ng.
- 1x DNA Dilution buffer: 2uL
Let the ligation sit 30 min. Then, using 3 vials of Top10F:
- 'c2' 19uL ligation product 30uL cells
- 'c2' 2uL ligation product 30uL cells
- 'c1 conc.' 21uL ligation product 30uL cells
- 'old' 19uL ligation product 30uL cells
- 'old' 2uL ligation product 30uL cells
- 'positive' The J04500 plasmid Hetmann miniprepped @21uL, 15uL of cells
- 'negative' 21uL water ,15uL cells.
30 min ice, 42C@30s heat, 2min ice, ~80min SOC, plating all on KAN except for the positive control, which is on CARB. Ready around 4pm.
Transformant plates
As of 12:30AM on 2006-8-24, there are still no colonies on the plates. I inoculated 4 cultures of the positive control we used (J04500 from Hetmann's midiprep), in 5 mL LB Amp, with 5 uL IPTG added.
