Team:INSA Toulouse/contenu/lab practice/notebook/protocols/comp cells





Competent Cells (iGEM Protocol)

This protocol is a variant of the Hanahan protocol using CCMB80 buffer for DH10B, TOP10 and MachI strains. It builds on Example 2 of the Bloom05 patent as well. This protocol has been tested on NEB10, TOP10, MachI and BL21(DE3) cells. See OWW Bacterial Transformation page for a more general discussion of other techniques. The Jesse '464 patent describes using this buffer for DH5α cells. The Bloom04 patent describes the use of essentially the same protocol for the Invitrogen Mach 1 cells. This is the chemical transformation protocol used by Tom Knight and the Registry of Standard Biological Parts.

· Detergent-free, sterile glassware and plasticware (see procedure)
· Table-top OD600nm spectrophotometer

CCMB80 buffer
· 10 mM KOAc pH 7.0 (10 ml of a 1M stock/L)
· 80 mM CaCl2.2H2O (11.8 g/L)
· 20 mM MnCl2.4H2O (4.0 g/L)
· 10 mM MgCl2.6H2O (2.0 g/L)
· 10% glycerol (100 ml/L)
· adjust pH DOWN to 6.4 with 0.1N HCl if necessary
adjusting pH up will precipitate manganese dioxide from Mn containing solutions.
· sterile filter and store at 4°C
· slight dark precipitate appears not to affect its function

Eliminating detergent
Detergent is a major inhibitor of competent cell growth and transformation. Glass and plastic must be detergent free for these protocols. The easiest way to do this is to avoid washing glassware, and simply rinse it out. Autoclaving glassware filled 3/4 with DI water is an effective way to remove most detergent residue. Media and buffers should be prepared in detergent free glassware and cultures grown up in detergent free glassware.

Prechill plasticware and glassware
Prechill 250mL centrifuge tubes and screw cap tubes before use.

Preparing seed stocks
· Streak TOP10 cells on an SOB plate and grow for single colonies at 23°C
room temperature works well
· Pick single colonies into 2 ml of SOB medium and shake overnight at 23°C
room temperature works well
· Add glycerol to 15%
· Aliquot 1 ml samples to Nunc cryotubes
· Place tubes into a zip lock bag, immerse bag into a dry ice/ethanol bath for 5 minutes
This step may not be necessary
· Place in -80°C freezer indefinitely.

Preparing competent cells
· Ethanol treat all working areas for sterility.
· Inoculate 250 ml of SOB medium with 1 ml vial of seed stock and grow at 20°C to an OD600nm of 0.3. Use the "cell culture" function on the Nanodrop to determine OD value. OD value = 600nm Abs reading x 10
This takes approximately 16 hours.
➔ Controlling the temperature makes this a more reproducible process, but is not essential.
➔ Room temperature will work. You can adjust this temperature somewhat to fit your schedule
➔ Aim for lower, not higher OD if you can't hit this mark

· Fill an ice bucket halfway with ice. Use the ice to pre-chill as many flat bottom centrifuge bottles as needed.
· Transfer the culture to the flat bottom centrifuge tubes. Weigh and balance the tubes using a scale
Try to get the weights as close as possible, within 1 gram.
· Centrifuge at 3000g at 4°C for 10 minutes in a flat bottom centrifuge bottle.
Flat bottom centrifuge tubes make the fragile cells much easier to resuspend · Decant supernatant into waste receptacle, bleach before pouring down the drain.
· Gently resuspend in 80 ml of ice cold CCMB80 buffer
Pro tip: add 40ml first to resuspend the cells. When cells are in suspension, add another 40ml CCMB80 buffer for a total of 80ml
➔ Pipet buffer against the wall of the centrifuge bottle to resuspend cells. Do not pipet directly into cell pellet!
➔ After pipetting, there will still be some residual cells stuck to the bottom. Swirl the bottles gently to resuspend these remaining cells

· Incubate on ice for 20 minutes
· Centrifuge again at 3000G at 4°C. Decant supernatant into waste receptacle, and bleach before pouring down the drain.
· Resuspend cell pellet in 10 ml of ice cold CCMB80 buffer.
If using multiple flat bottom centrifuge bottles, combine the cells post-resuspension
· Use Nanodrop to measure OD of a mixture of 200 μl SOC and 50 μl of the resuspended cells
Use a mixture of 200 μl SOC and 50 μl CCMB80 buffer as the blank
· Add chilled CCMB80 to yield a final OD of 1.0-1.5 in this test.
· Incubate on ice for 20 minutes. Prepare for aliquoting
Make labels for aliquots. Use these to label storage microcentrifuge tubes/microtiter plates
➔ Prepare dry ice in a separate ice bucket. Pre-chill tubes/plates on dry ice.

· Aliquot into chilled 2ml microcentrifuge tubes or 50 μl into chilled micro titer plates
· Store at -80°C indefinitely.
Flash freezing does not appear to be necessary
· Test competence (see below)
· Thawing and refreezing partially used cell aliquots dramatically reduces transformation efficiency by about 3x the first time, and about 6x total after several freeze/thaw cycles.

Measurement of competence
· Transform 50 μl of cells with 1 μl of standard pUC19 plasmid (Invitrogen)
This is at 10 pg/μl or 10-5 μg/μl
➔ This can be made by diluting 1 μl of NEB pUC19 plasmid (1 μg/μl, NEB part number N3401S) into 100 ml of TE

· Incubate on ice 0.5 hours. Pre-heat water bath now.
· Heat shock 60 sec at 42C
· Add 250 μl SOC
· Incubate at 37 C for 1 hour in 2 ml centrifuge tubes, using a mini-rotator
Using flat-bottomed 2ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration.
➔ For our plasmids (pSB1AC3, pSB1AT3) which are chloramphenicol and tetracycline resistant, we find growing for 2 hours yields many more colonies
➔ Ampicillin and kanamycin appear to do fine with 1 hour growth

· Add 4-5 sterile 3.5mm glass beads to each agar plate, then add 20 μl of transformation
After adding transformation, gently move plates from side to side to re-distribute beads. When most of transformation has been absorbed, shake plate harder
➔ Use 3 plates per vial tested

· Incubate plates agar-side up at 37 C for 12-16 hours
· Count colonies on light field the next day
Good cells should yield around 100 - 400 colonies
➔ Transformation efficiency is (dilution factor=15) x colony count x 105/µgDNA
➔ We expect that the transformation efficiency should be between 1.5x108 and 6x108 cfu/µgDNA

5x Ligation Adjustment Buffer
· Intended to be mixed with ligation reactions to adjust buffer composition to be near the CCMB80 buffer
· KOAc 40 mM (40 ml/liter of 1 M KOAc solution, pH 7.0)
· CaCl2 400 mM (200 ml/l of a 2 M solution)
· MnCl2 100 mM (100 ml/l of a 1 M solution)
· Glycerol 46.8% (468 ml/liter)
· pH adjustment with 2.3% of a 10% acetic acid solution (12.8ml/liter)
Previous protocol indicated amount of acetic acid added should be 23 ml/liter but that amount was found to be 2X too much per tests
· water to 1 liter
· autoclave or sterile filter
· Test pH adjustment by mixing 4 parts ligation buffer + 1 part 5x ligation adjustment buffer and checking pH to be 6.3 - 6.5