Glycerol Stock

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.