Jonesma Lab:Protocols:Agrotransform: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
(New page: Thaw competent Agrobacterium on ice (use 250 ul per transformation reaction) and add DNA (1-5 ul of CsCl-purified DNA or 10 ul of standard E. coli miniprep DNA). Incubate the mixture on i...) |
No edit summary |
||
| Line 1: | Line 1: | ||
<h3><font style="color:#F8B603;">Transforming Agrobacteria with cold-shock transformation)</font></h3> | |||
---- | |||
Incubate the mixture on ice for 5 minutes | #Thaw competent Agrobacterium on ice (use 250 ul per transformation reaction) and add DNA (1-5 ul of CsCl-purified DNA or 10 ul of standard E. coli miniprep DNA). | ||
#Incubate the mixture on ice for 5 minutes | |||
Transfer mixture to liquid nitrogen and incubate for 5 minutes. | #Transfer mixture to liquid nitrogen and incubate for 5 minutes. | ||
#Incubate the mixture for a further 5 minutes in a 37 degree C water bath. | |||
Incubate the mixture for a further 5 minutes in a 37 degree C water bath. | #Add 1 ml of LB to each tube, seal well, and place the tubes on a rocking table for 2-4 hours at room temperature. | ||
#Collect the cells by spinning briefly in a microcentrifuge and spread them on two LB agar plates containing the appropriate antibiotic. Include the antibiotic for the T-DNA vector. | |||
Add 1 ml of LB to each tube, seal well, and place the tubes on a rocking table for 2-4 hours at room temperature. | #Incubate cells for 2 days at 28 degrees C. | ||
Collect the cells by spinning briefly in a microcentrifuge and spread them on two LB agar plates containing the appropriate antibiotic. Include the antibiotic for the T-DNA vector. | |||
Incubate cells for 2 days at 28 degrees C. | |||
Latest revision as of 03:00, 27 July 2015
Transforming Agrobacteria with cold-shock transformation)
- Thaw competent Agrobacterium on ice (use 250 ul per transformation reaction) and add DNA (1-5 ul of CsCl-purified DNA or 10 ul of standard E. coli miniprep DNA).
- Incubate the mixture on ice for 5 minutes
- Transfer mixture to liquid nitrogen and incubate for 5 minutes.
- Incubate the mixture for a further 5 minutes in a 37 degree C water bath.
- Add 1 ml of LB to each tube, seal well, and place the tubes on a rocking table for 2-4 hours at room temperature.
- Collect the cells by spinning briefly in a microcentrifuge and spread them on two LB agar plates containing the appropriate antibiotic. Include the antibiotic for the T-DNA vector.
- Incubate cells for 2 days at 28 degrees C.
