User:Tk/Notebook/MF-xfm/2008/04/25: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
(Autocreate 2008/04/25 Entry for User:Tk/Notebook/MF-xfm) |
|||
| Line 5: | Line 5: | ||
|- | |- | ||
| colspan="2"| | | colspan="2"| | ||
== | ==Transformations== | ||
* | * Transform 400 ul sample frozen at -80 | ||
* Thaw on ice | |||
* Add 16 ul transposome (4/24) | |||
* Add 80 ul 1 M sorbitol | |||
* Dispense 4x into 1 mm chilled cuvettes | |||
* Hold on ice 10 min | |||
* Electroporate at 1.5 KV | |||
* Immediately add 1 ml 1161 medium | |||
* Hold on ice 10 min | |||
* Incubate in the cuvette for 50 minutes at 30C | |||
* Plate out 330 ul onto 12x 1161 + tet plates | |||
==More transposon construction== | |||
* Grow pTT01 plasmid (10 ml overnight culture + 500 ml LB/Kan in a shake flask) | |||
* Harvest in conical centrifuge tubes after 9 hour growth | |||
* Discard supernatent, freeze pellet | |||
==PCR transposon construction== | |||
* 100 ul PCR reaction | |||
** 50 ul Phusion m/m | |||
** 47 ul water | |||
** 3 ul ME0 primer | |||
** cycle 98/30s (98/15s 55/15s 68/1:15 min)x36 | |||
<!-- ## Do not edit below this line unless you know what you are doing. ## --> | <!-- ## Do not edit below this line unless you know what you are doing. ## --> | ||
|} | |} | ||
Revision as of 21:54, 19 June 2008
 MF-xfm
|
<html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> |
Transformations
More transposon construction
PCR transposon construction
| |
