Plasmid Transformation and Isolation (Appendix A)

(LB) AGAR

(Invitrogen 22700-025)

            32.0 gm per liter of MilliQ water

            Autoclave at 121°C for 15 min

 

(LB) Broth

(Fisher BP1426-500)

Suspend 25g in 1 L of MilliQ water

 

•Place in 500ml dH2O (Makes approx. 20 plates)

• Adjust pH 7.5 then autoclave

(Note: Agar does not dissolve into soln until autoclaved)

• Remove LB from autoclave & place in 50°C H2O bath immediately until use.

 

To Make LB Agar Plates

  1. Clean countertop surface w/ 70% ETOH
  2. Add 1ml Ampicillin (AMP 10mg/ml) per 100ml LB Agar. (Heat top of AMP tube to sterilize) (0.1mg/ml final)
  3. Pour LB Agar into Petri dishes (Falcon IC) making approx. 20 plates.
  4. Let harden at RT.

 

To Grow Up Plasmid

  1. Dilute supercoiled stock to concentration of 100ng/10µl. Prepare approx. 20µl of each dilution.
  2. Remove cells (Invitrogen DHSa) from -70°c; thaw on wet ice.
  3. Place tubes on wet ice (Kaunitz’ lab has 14ml plastic sterile tubes) & label.
  4. Gently mix cells
  5. Aliquot 100µl/tube (3ml Falcon sterile culture tubes)
  6. Add 20µl of desired cDNA dilution (100ng/10µl) to the 100µl of bacterial cells already aliquoted. (Heat top of tube to sterilize)

-One tube is for transformation efficiency. Add 5µl of “control” DNA to one of the tubes aliquoted.

  1. Incubate on ice for 30’
  2. Heat shock cells 45 seconds in a 42°c water bath. Do not shake. Time is very important.
  3. Place on ice for 2 minutes.
  4. Add 0.9ml of LB broth medium to each tube.
  5. Shake @ 37°c for 1 hour.
  6. At this point, dry plates that were made earlier in fume hood for approx. ½ hour or until (remove lid pt) condensation of top of lids are dry.
  7. Next, before spreading bacterial cells onto agar plates, wipe down countertop w/ 70% Etoh and place glass pipets into Etoh in beaker to soak.
  8. Now, to spread cells onto plates A) Remove lid B) pipet 20µl (or 50µl) of cells onto agar plates C) place lid back onto plate D) take glass pipet from Etoh and heat w/ flame (Bunsen burner) to sterilize and bend end E) cool pipet tip by placing on surface of agar (where no cells are located) F) spread cells over plate.
  9. Incubate plates upside down @ 37°c at clinic O/N.

 

Next Day

  1. Remove plates to 4°c. There should be a good number of visible colonies. These indicate bacteria that have ampicillin resistance which is the result of transformation w/ a plasmid w/ Ampr sequence.
  2. Make 1 liter of broth (LB medium w/ NO AGAR); separate into 4 1L flasks (those with metal caps) (need 4x volume in flasks as volume of LB [250 ml in 1L flask])  and autoclave. Remove to 4°c.  (Can make ahead)
  3. Add 2.5 ml Ampicillin (10mg/ml) (1ml/100ml) to one of the flasks containing 250ml of broth (@<50°c) (add amp. immediately before use)
  4. Add 5ml to small vial (14ml vial)
  5. Select 1 colony that is circular and isolated, and touch sterile wand to it.
  6. Swoosh wand around in 5ml broth vial (heat with flame)
  7. Incubate 4.5 hours @ 37°c shaking. If needed, let incubate o\n and do miniprep at this point to verify identity. (Qiagen miniprep kit)
  8. Add 2.5 ml Ampicillin to each of the 3 remaining flasks
  9. Add 0.5 ml of the 5ml broth vial to each of the 4 (250 ml) flasks (heat w/ flame)
  10. Incubate 4 flasks @ 37°c rotating O/N. (can freeze some of this culture 1:1 glycerol. Freeze on dry ice)

 

Day 3

  1. Flasks should be cloudy
  2. Place contents of flasks into large centrifuge bottles
  3. Centrifuge @ 7000rpms for 30’ @ 4°c (Note: Beforehand cool down centrifuge to 4°c)
  4. Remove supernatant and toss (you get a big pellet)

 

 

For Plasmid maxi preps refer next to Qiagen protocol (at end of this protocol)