Team:Hawaii/Initial E. Coli Transformation

= Transformation of DH5-alpha =

Materials
Materials:


 * 37°C shaking and non-shaking incubator
 * 10 cm diameter LB agar plates with appropriate antibiotic (100 µg/ml ampicillin to select transformants containing pUC19 control DNA)
 * LB, YT, or SOC Medium
 * Dry ice and ethanol
 * 37°C water bath

Before Starting

 * Prepare a dry ice/ethanol bath and maintain at -70°C
 * Equilibrate a water bath to 37°C
 * Spread X-Gal onto LB agar plates with antibiotic, if desired
 * Warm the medium and plates in a 37°C incubator for 30 minutes
 * Obtain a test tube rack that will hold all transformation tubes so that they can all be put into a 37°C water bath at once.

Transformation Procedure
Note: Plasmid DNA should be free of phenol, ethanol, protein, and detergents for maximum transformation efficiency.


 * 1) Briefly centrifuge the ligation reaction and place on wet ice.
 * 2) Remove one 500 µl tube of DH5α™ cells and thaw on wet ice.
 * 3) Place the required number of sterile 1.5 ml microcentrifuge tubes on wet ice.
 * 4) Gently mix cells with the pipette tip and aliquot 50 or 100 µl into each microcentrifuge tube.
 * 5) Re-freeze any unused cells in the dry ice/ethanol bath for 5 minutes before returning the tube to the -70°C freezer.
 * 6) Pipet 1 to 5 µl (1-10 ng DNA) of each ligation reaction directly into the competent cells and mix by tapping gently. Do not mix by pipetting up and down. Store the remaining ligation reaction at -20°C.
 * 7) (Optional) Pipet 5 µl (500 pg) pUC19 control DNA into 100 µl competent cells and mix as described in Step 6.
 * 8) Incubate the vial on ice for 30 minutes.
 * 9) Heat-shock for exactly 20 seconds in the 37°C water bath for 50 µl volume (45 seconds for 100 µl transformation). Do not mix or shake.
 * 10) Remove vial from the 37°C bath and place on ice for 2 minutes.
 * 11) Add 900 to 950 µl of pre-warmed medium of choice to each vial. Proceed to next page.
 * 12) Place the vial in a microcentrifuge rack on its side and secure with tape to avoid loss of the vial. Shake the vial at 37°C for exactly 1 hour at 225 rpm in a shaking incubator.
 * 13) Spread 20 µl to 200 µl from each transformation vial on separate, labeled LB agar plates. We recommend that you plate two different volumes. You may have to dilute cells 1:10 to obtain well-spaced colonies. Generally ligations are at least 10-fold lower efficiency.
 * 14) (Optional) For cells transformed with pUC19 control DNA, plate 100 µl of the transformation reaction, then serially dilute the transformation reaction 1:10 and 1:100 and plate 100 µl of each dilution on plates containing 100 µg/ml ampicillin.
 * 15) Store the remaining transformation reaction at 4°C and plate out the next day, if desired. If necessary, cells may be concentrated by centrifuging in a microcentrifuge (5 seconds) and resuspending them in 100 µl. Plate 1 to 90 µl.
 * 16) Invert the plates and incubate at 37°C overnight.

Reference
"Subcloning Efficiency™ DH5α™ Competent Cells." 2006. Invitrogen. 01 June 2008. Available http://tools.invitrogen.com/content/sfs/manuals/subcloningefficiencydh5alpha_man.pdf.

Protocol from SC Lab

 * 1) Thaw cells on ice
 * 2) Add 10 µl ligation reaction with a pipetter and mix by flicking the tube gently
 * 3) Incubate the tube 30 minutes on ice
 * 4) Heat shock cells for 2 minutes at 42º C. To do this, transfer the cells directly from ice to a water bath at 42º C.
 * 5) Incubate on ice for 2 minutes
 * 6) Add 800 µl SOC media and transfer to a test tube
 * 7) Shake at 37º C for 2.5 hours (30 min is OK for Ampicillin resistance)
 * 8) Plate 200 µl of cells on selective media.

Questions

 * 1) Concentration of plasmid used?
 * 2) How much cells?

Protocol Used

 * 1) Thawed cells (2-17, OD600 = 0.3) on ice.
 * 2) Transformed 50 &mu;l cells w/ 1 &mu;l pRL1383a plasmid (10 pg per &mu;l)
 * 3) Incubated on ice 30 min.
 * 4) Heat shocked 60 sec. in 42C water bath
 * 5) Added 250 &mu;l SOC
 * 6) Incubated @ 37C for 1 hour with shaking (235 rpm)
 * 7) Plated 20 &mu;l onto LB+spectinomycin plates

Results
80 colonies were observed 16 hours after innoculation. Transformation efficiency = 4 transformants/&mu;l

Discussion
= Transformation of DB3.1 Competent Cells =

Attempts 1-3



Attempt 4

 * I had chosen the wrong plasmids, so I did another plasmid prep that is destined to win because of all my experience!