IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-12: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
No edit summary |
JeffreyLau (talk | contribs) No edit summary |
||
Line 70: | Line 70: | ||
[[Image:ligationtest_ZS.jpg|thumb|left]] | [[Image:ligationtest_ZS.jpg|thumb|left]] | ||
[[Image:2006-08-12 Cyano Digest Gel Failure.jpg|thumb|left]] | [[Image:2006-08-12 Cyano Digest Gel Failure.jpg|thumb|left]] | ||
==Redigest of Kai\X-P== |
Revision as of 18:46, 12 August 2006
<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>
To-do
RFP in low-copy plasmid (transformed in Top10 and Top10F')- GFP dev in low-copy plasmid (from digest assay)
Run the redigest of pSB4A3 (pSB4A3\S-P, pSB4A3\X-P, psB4A3 undigested) on a gel, to see if the plasmid is the right length or if it has an insertOur gel boiled in the tank; we've labeled the tank armed and dangerous. We lost this DNA.- CIP treat the 4 gel-extracted pSB4A3\S-P from the digest assay
- If the pSB4A3 plasmid looks alright, ligate GFP dev\X-P + psB4A3\S-P
- Transform ligation products
- Kai\X-P + J04500\S-P ligation
Run the KaiA\X-P, KaiB\X-P, and KaiC\X-P digests from yesterday on a gelOur gel boiled in the tank; we've labeled the tank armed and dangerous. We lost this DNA.Redo the KaiA\X-P, KaiB\X-P, and KaiC\X-P digests- Run the KaiA\X-P, KaiB\X-P, and KaiC\X-P digests on a gel
- Extract and purify KaiA\X-P, KaiB\X-P, and KaiC\X-P
- CIP treat J04500\S-P
- Ligate Kai\X-P + J04500\S-P
- Transform ligation products
Growing up Kai--Zsun 17:37, 12 August 2006 (EDT)Inoculate cultures of the transformed KaiA/B/C GeneART plasmids
- More backbone
- Grow up a lot of pSB4A3- and J04500- transformed cells for midiprepping
Results of Restreak of J00450+PSB4A3
Although the colonies took two days to grow, the restreak of the plates on Carb worked; we will now verify the ligation via PCR while growing liquid culture from them.
PCR ligation test of J04450+PSB4A3 + innoculation
Following Perry's Protocol which worked pretty well, in order to check the ligation we will do a PCR using the PCR Supermix from the VF2 and VR areas of the plasmid;
From the above image, we will try 7 colonies:
- Top10 plate2 A (eg. 102)
- Top10 plate1 A (eg. 101)
- Top10F plate1 (eg. F1)
- Top10F plate2 1 (eg. F2-1)
- Top10F plate2 2 (eg. F2-2)
- Top10F plate2 3 (eg. F2-3)
- Top10F plate2 3 (eg. F2-4)
From Perry's Protocol, each will have:
- 8uL PCR supermix
- 1uL VF2 (2uM)
- 1uL VR (2uM)
Then, get a carb plate, pick the desired colony, grow in 8mL LB+Carb. Choose another (monoclonal source), dip it into the 10uL rxn.
The runtime protocol, modified from Perry's is:
- 95@15m
- Loopx30
- 95@30
- 55@30
- 72@1.30
- 72@10
- 4@forever