Category Archives: Lab Protocol

PROTOCOL: Thawing FBS and Storing FBS

  1. Remove the serum bottles from the freezer and allow them to acclimate to room temperature for approximately 10 minutes

  2. Place each container in a 30 to 37°C water bath or incubator. Excessive temperatures will degrade heat-labile nutrients. Prevent the bottle from tilting over.

  3. Gently swirl or shake the bottles every 10 to 15 minutes until is completely thawed.

  4. After thawing, use serum promptly. Liquid serum may be stored refrigerated (2 to 8°C) for up to 4 weeks. Ultimately, this length of time where cell culture can be successful will vary with different cell lines. Repeated freeze and thaw series may cause a decrease in serum performance. To avoid thaw/freeze cycles or long periods of refrigeration it is recommended that any unused serum be immediately dispensed into small aliquots and refrozen.

Adapted from: http://www.biowestusa.com/

Mini-prep without column

To save up money, you can do mini-prep without columns. How pure and how much more time do you spend though? The answers are 1) very pure; 2)  25 more minutes.

The protocol is adapted from Gomez lab. This should work with any reagents for Mini-prep.

1. Spin down bacterial culture for 1 minute (13-14k rpm)

2. Aspirate supernatant

3. Resuspend pellet with 200ul resuspension buffer

4. Lyse the bacteria with 250ul lysis buffer

5. Precipitate protein with 350ul precipitation buffer

6. Spin for 10 minutes

7. Take out supernatant to a new tube

8. Top up with ethanol. You should see DNA precipitate, ie. the solution gets cloudy

 

(picture: cloudy solution)

9. Spin down at 14k rpm for 30 minutes

10. Aspirate supernatant, add 500ul of 70% ethanol, Spin down at 14k rpm for 1mininute

11. Aspirate supernatant, open the cap and let it dry for 5 minutes

12. Resuspend in 40ul of TE buffer

This protocol is surprisingly clean. There is no need to add RNase A to get rid of RNA. I didn’t see any smear on gel.

(left to right: marker, lane4=undigested vector, lane5=digested vector)

Adapted from: http://openwetware.org/wiki/Jimenez-Gomez_Lab:protocol_miniprep_without_columns