Protein Expression and Purification Series - Bio-Rad

Protein Expression and Purification Series
Tasks to Perform Prior to the Lab
Note: Several reagents are used in different parts of the advance preparation as well as for different
laboratory activities. Do not discard excess reagents used in preparation and store them at the
appropriate conditions.
Streaking starter plates to produce single bacterial colonies on agar plates
1. Preparation of ampicillin: With a sterile pipet, add 600 µl of sterile water to each vial containing
30 mg ampicillin to rehydrate the antibiotic, making a 50 mg/ml (500x) solution.
Note: Ampicillin is used to make LB/amp agar plates as well as LB/amp broth, so do not discard any
unused ampicillin solution. The rehydrated ampicillin should be stored at –20°C and is good for one
year.
2. Preparation of LB/amp agar plates: At least two days prior to cell culturing, prepare one LB/amp
agar plate per team to be used to inoculate E. coli starter cultures.
Note: Only prepare 200 ml of molten LB agar reagent (enough for 20 plates) to which 200 µl of 50
mg/ml ampicillin will be added to make LB/amp agar plates. Retain excess 50 mg/ml ampicillin at
–20°C for use in preparation of LB/amp broth.
This protocol is used to prepare solid LB/amp agar media to prepare plates for the growth of starter
colonies of BL21(DE3) E. coli containing the pDHFR plasmid. Each student group will require one LB/amp
agar plate. Agar plates should be prepared at least two days before required, left out at room temperature
for two days and then refrigerated until they are to be used. Two days on the bench allows the agar to dry
out and more readily take up the rehydrated E. coli solution.
Figure 3B.1. Preparation of LB/amp agar.
A. To prepare 20 LB/amp plates, prepare 200 ml of LB agar. Add 8 g of LB nutrient agar to 200 ml
of distilled water in a 500 ml or larger Erlenmeyer flask and cover the flask opening. Swirl the flask
to dissolve the agar, or add a magnetic stir bar to the flask and stir on a stir plate. A stir bar will also
aid in mixing the solution once sterilization is complete. Autoclave the LB agar on the wet cycle for
30 minutes. Once the autoclave cycle is complete, check the solution to ensure that the agar is
completely dissolved.
Note: Be sure to wear appropriate protective equipment and be careful to allow the flask to cool
slightly before swirling process so that the hot medium does not boil over onto your hand.
If no autoclave is available, heat the agar solution to boiling in a microwave or on a hot plate. Repeat
the heating and swirling about three times until all the agar is dissolved (no more clear specks are
noticeable). Use a lower power setting on the microwave to reduce evaporation and boilover.
Chapter 3B: Advance Preparation for
Chromatography Instrumentation Protocols
73
CHAPTER 3B
ADVANCE PREP
INSTRUMENTATION PROCES