From 2008.igem.org

Latest revision as of 02:31, 2 August 2008

Transformation of DH5-alpha

Protocol by Invitrogen

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.

Notes

1. Transformation efficiency should be greater than 1 x 106 transformants/µg pUC19 DNA.
2. To select transformants using blue/white screening, make sure that selective plates contain 50 µg/ml X-gal.
3. Concentration of pUC19 provided in Invitrogen kit is 100 pg/µl.

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.

Notes

1. Add 10 ml sterile 1M MgCl2 and 10 ml 1M MgSO4 to SOB media before use.

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 μl cells w/ 1 μl pRL1383a plasmid (10 pg per μl)
3. Incubated on ice 30 min.
4. Heat shocked 60 sec. in 42C water bath
5. Added 250 μl SOC
6. Incubated @ 37C for 1 hour with shaking (235 rpm)
7. Plated 20 μl onto LB+spectinomycin plates

Results

80 colonies were observed 16 hours after innoculation. Transformation efficiency = 4 transformants/μl

Transformed DH5α on LB+sp

Discussion

Transformation of DB3.1 Competent Cells

Attempts 1-3

Transformation of DB3.1

Attempt 4

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

Colony PCR: verified plasmids pSB1A3, pSB1A2, pSB3K3, and additionally verifying a sample of pSMC121 graciously donated by Dr.C.

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