Paul.xu: Created page with "1. Add 5ml LB+ antibiotics to 15 ml culture tube.
2. Pick up one single colony using 10 ul tip. DO not place tip inside the culture tube.
3. Grow in a shaker incubator ..."

2012-09-01T02:23:23Z

Created page with "1. Add 5ml LB+ antibiotics to 15 ml culture tube. 2. Pick up one single colony using 10 ul tip. DO not place tip inside the culture tube. 3. Grow in a shaker incubator ..."

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1. Add 5ml LB+ antibiotics to 15 ml culture tube. 

2. Pick up one single colony using 10 ul tip. DO not place tip inside the culture tube. 

3. Grow in a shaker incubator at 37 oC o/n. 

4. Next morning, take 10 ul out from the culture tube to a new 1.5 ml tube, add 0.5 ml LB+antibiotics medium. 

5. Spin down cells in 15 ml tube, make miniprep DNA using home made buffers and silica spin  column. 

6. After a positive is identified (by restriction digestion, PCR, or sequencing), grow the positive clone bacteria  in 5 ml LB+ antibiotics in 15 ml culture tube. 

7. Add 0.5 ml autoclaved 80% glycerol to 1.5 ml screw cap tube. 

8. Spin down E coli. cells, resuspend cells in 0.5 ml medium. Transfer cells to the screw cap tube. 

9. Print a freezer label with these infomation:

* Clone number

* Clone name

* Host (e.g., DH5alpha, Top10, STBL3, ccdB Survival etc)

* Antibiotics, A for amp, K for Kan

* If it is sequenced, label it as Seq.

* Date.

10. Label the tube with the label, write clone number on top of the tube.

11. Store tube in a freezer box.

12. Update clone information on clone database.

[[Category:Protocols]]

Glycerol Stock - Revision history

Paul.xu: Created page with "1. Add 5ml LB+ antibiotics to 15 ml culture tube. 2. Pick up one single colony using 10 ul tip. DO not place tip inside the culture tube. 3. Grow in a shaker incubator ..."

Paul.xu: Created page with "1. Add 5ml LB+ antibiotics to 15 ml culture tube.
2. Pick up one single colony using 10 ul tip. DO not place tip inside the culture tube.
3. Grow in a shaker incubator ..."