Preparation of Bacterial Freezer Stocks

Preparation:

1. Take Amp out of freezer to thaw. 
2. Take Freezer Stock streak plates out of fridge. 
3. Label 50mL blue cap tubes with Sample ID. 
4. Label two sets of cryo-freezing tubes with Sample ID and date 
               a.    Sample ID example: GTL10001, APC10012

Preparation:

1. Inoculate 15 ml of LB/antibiotic medium (in a 50ml blue-cap tube) with bacteria from a fresh plate culture. 
2. Incubate at 37°C for 4 hours until an OD600 of 1-2 is reached (the OD does not have to be measured. When the medium is cloudy, when looking through the back side can't see numbers through the tube). 
3. Collect the bacteria by centrifugation at 2,500 x g for 10 minutes 
4. While centrifuging samples prepare LB/DMSO solution. LB plus 8% DMSO by volume (i.e. 21 samples would require 21ml of LB plus 1.68μl of DMSO). 
5. Discard the supernatant and resuspend the pellet in 1 ml of LB/DMSO. 
6. Transfer the bacterial suspension into two cryo-freezing tubes and place on dry ice 
7. Store the frozen stocks at -70-190°C 
8. Enter the freezer location and bacterial stock data in the freezer log