Team:Caltech/Week 6
From 2011.igem.org
Line 89: | Line 89: | ||
Gel extraction of 40kb LA River sample DNA from pulse field gel<br/> | Gel extraction of 40kb LA River sample DNA from pulse field gel<br/> | ||
Transform pSB3K3 into competent cells, since the pSB3K5 transform is not working<br/> | Transform pSB3K3 into competent cells, since the pSB3K5 transform is not working<br/> | ||
+ | Run Gibson assembly of Joe's GFP and pNT001 and stop at different time points. Run on a gel to see what size DNA there is at different times in the reaction<br/> | ||
+ | Try "step-wise" Gibson. Add the insert for pNT001 (3 ul) into a 10 ul reaction and incubate at 50˚C for 10 or 20 minutes before adding to a premade Gibson mix of just the vector (2 ul). Continue incubating until a total of 1 hour has passed. This may help with the self-ligation of the vector by giving the insert time to assemble if it works<br/> | ||
===Results=== | ===Results=== | ||
Gibson Plates | Gibson Plates | ||
Line 113: | Line 115: | ||
</tr> | </tr> | ||
</table> | </table> | ||
+ | [[File:7-21gibsontime.jpg|thumb|200px|left|lane 1 NEB 2-log ladder, 2 GFP 0 min, 3 GFP 5 min, 4 GFP 10 min, 5 GFP 15 min, 6 GFP 30 min, 7 GFP 60 min, 8 blank, 9 pNT001 0 min, 10 pNT001 5 min, 11 pNT001 10 min, 12 pNT001 15 min, 13 pNT001 30 min, 14 pNT001 60 min, 15 NEB 2-log ladder. Triangles represent increasing time (not to scale)]] | ||
}} | }} |
Revision as of 06:42, 22 July 2011
Project |
July 18Colony PCR of colonies from positive Gibson plates from Friday, for vector and insertColony PCR of 16s plasmids, for vector and insert ResultsGels show mostly expected single bands July 19PCR parts for Gibson assembly of pNT001, pNT003 and Joe's positive GFP control ResultspSB3K5 plate had 0 colonies. Gibson PCR concentrations
July 20Pulse field gel of soil-extracted DNA, in preparation for fosmid insertion ResultsPCR concentrations
July 21Gel extraction of 40kb LA River sample DNA from pulse field gel ResultsGibson Plates
|