NS5A was purified from Top10 E. coli. The concentration was not desirable. The protocol will be repeated with more time for the E. coli to reach mid log phase growth.
Project: Regulation of Phosphotidylinositol-4-phosphate by Ttc7 and its role in plasma cell differentiation and autoantibody production.
Ongoing- First attempted July 2014
To propogate plasmids in E. Coli for use in the Pelsue Laboratory.
Prepare the following:
LB Broth- 20 g/L
Add 20 g of LB broth powder to 1L of water. Autoclave on cycle 6
LB Agar- 32g/L
Add 32 g of LB agar powder to 1L of water. Autoclave on cycle 6
Prepare antibiotic stocks:
Ampicillin- use at 100 ug/mL
Stock- AMP 50 mg/mL (=50 ug/uL)
[50mgX10mL= 500 mg in 10 mL sterile water]
Kanamycin- use at 30 ug/mL
Stock- 15 mg/mL (=15 ug/uL)
[15mgX10 mL= 150mg in10 mL sterile water]
Once prepared the above antibiotics will used to add to media and to broth as necessary.
Prep cloning room:
EtOH, bleach, Gloves, paper towels, Petri dishes (label LB Agar, date, MS, antibiotic [conc])
Add antibiotic to autoclaved agar in the cloning room. Make sure the agar has cooled enough to touch but not too cool either.
Add 1 mL of prepared antibiotic to 500 mL agar. Invert to mix.
Plate 25 mL of agar+antibiotic mixture to each labelled petri dish. Let sit to dry with cap tipped to let out condensation. Let solidify. Once cooled and dry wrap in parafilm and store in refrigerator 3.
To streak plates:
Use prepared plates from above. Make sure ethanol burner is full of 200 proof ethanol. Streak plates in a four corner manner to allow individual colonies to grow. Streaking from stabs is very easy, just poke metal loop into stab and streak from that. Once streaked allow to dry shortly and place in 37 degree incubator overnight. If no individual colonies are present, restreak plates from original plates.
Prepare 15mL conical tubes with 5 mL of LB broth. Add antibiotics to broth beforehand. Add 10 uL of antibiotic to 5 mL of broth.
To select an individual colony circle the petri dish where the desired colony is, and poke with micropipetter. Transfer to 5 mL of antibiotic + broth in 15 mL conical tube. Put on shaker incubator for 6-9 hours at 37 degrees, 225 rpm. This is the starter culture.
From starter culture (once the bacteria appears to be in log growth, the media should be quite cloudy). Take 1 mL of starter culture and add to either 25 mL or 50mL of broth+ antibiotic. For 50 mL add 100 uL of antibiotic for 25 mL add 50 uL of antibiotic. Place culture back on shaker incubator at same settings for 24 hours.
Many things can be done to the bacterial cultures at the conclusion of the above protocol. To isolate and purify plasmid the Promega wizard plus miniprep kit is used. To make glycerol stocks the following protocol is used.
Label cryotubes ahead of time. Make a glycerol media ahead of time. Add antibiotics to media before use. The ratio for media to glycerol should be 85:15. Centrifuge the bacterial cultures at 3000 rpm, 4 degrees, for 7 min. Resuspend cell pellet in the glycerol media. Store the cryotubes in the -70 freezer. Each crytoube will hold 1 mL if 5 glycerol stocks are being prepared then 4.25 mL of media and 0.750 mL of glycerol will be used. Glycerol stocks are good for about 2 years.
If one does not have stabs to start from Top10 bacteria can be transformed using the plasmid of interest. To do so the Top10 Transforming Competent Cells protocol is used.
Results are gathered using the nanodrop
The nanodrop may not be particularly accurate. Sometimes the bacteria needs more time during the culturing period to get to log growth. Experiment times change depending on state of bacterial culture.
This protocol is very general. Different plasmids may require slight alterations.
Sample Storage Location:
Plasmids are in freezer 3