Jul 22, 2019
Preparing Chemically Competent E coli for Transformation
- 1University of California, San Francisco
- Stephen Floor Lab
Protocol Citation: Stephen Floor 2019. Preparing Chemically Competent E coli for Transformation. protocols.io https://dx.doi.org/10.17504/protocols.io.4pagvie
Manuscript citation:
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 24, 2019
Last Modified: July 22, 2019
Protocol Integer ID: 25026
Keywords: competent cells, bacteria, transformation, chemical competence
Abstract
This protocol prepares chemically competent E coli for plasmid transformation. The competency is high enough for routine cloning but working with libraries may require higher competency. The protocol has worked with every E. coli type I've tried it with. Frozen cells are stable for years at -80, and can be refrozen with minimal effects to their competency. Recommended to get a friend or two to help with aliquoting the bacteria at the end, both so that they stay cool (important) and so that you stay cool (important).
Adapted from Promega Cloning Manual Notes and Inoue/Hanahan protocol, DNA Cloning 1985. With help from Amy Lee, Brandeis (http://www.bio.brandeis.edu/leelab/).
Attachments
bakktw.pdf
42KB
Guidelines
Additional Protocol Notes
- Protocol works well to prepare cells with 1 plasmid already inside (Cam+tRNA RIL plasmid for example)
- Add selection to original plate and growth media, but do not add selection to TFB1/TFB2 buffers
- Can prepare plates with odd antibiotic by first plating antibiotic onto a LB agar plate
- Pre-mix enough antibiotic for 15 ml of LB into 100 µl of LB and evenly spread mixture onto the plate and allow to dry at RT for ~30 min before streaking cells.
Materials
TFB1 (Prepare 300 ml)
30 mM KOAc (98.15 g/mol) 0.883 g
10 mM CaCl2•2H2O (147.01 g/mol) 0.441 g
50 mM MnCl2•4H2O (197.91 g/mol) 2.969 g
100 mM RbCl (120.92 g/mol) 3.628 g
15% Glycerol 45 ml (100% glycerol stock)
dH2O 220 ml, qs to 290 ml
*pH to 5.8 with 1.75 M Acetic Acid 10–20 drops (SLOWLY)
- Dilute glacial acid 1/10 for use
- pH of starting solution is ~7.1
*qs to 300 mL with dH2O and 0.2 µm-filter sterilize
TFB2 (Prepare 300 ml)
100 mM MOPS (209.26 g/mol) 6.278 g
75 mM CaCl2•2H2O (147.01 g/mol) 3.308 g
10 mM RbCl (120.92 g/mol) 0.363 g
15% Glycerol 45 ml (100% glycerol stock)
dH2O 220 ml, qs to 290 ml
*pH to 6.5 with 2 M KOH 2–4 ml (SLOWLY)
- 2 M KOH: 1.122 g (56.11 g/mol) in 10 mL dH2O
- pH of starting solution is ~4.2
*qs to 300 mL with dH2O and 0.2 µm-filter sterilize
1 M MgSO4 (Prepare 45 ml): Add 11.09 g MgSO4•7H2O (246.47 g/mol) to 40 ml, qs to 45 ml and 0.2 µm-filter sterilize
Safety warnings
See SDS (Safety Data Sheet) for hazards and safety warnings.
Before start
For Competent Cell Preparation
- Be prepared to work in 4°C room.
- Pre-chill buffers TFB1 and TFB2 on ice.
- Pre-chill p1000 tips at 4°C.
- Label and pre-chill 1.5 mL microfuge tubes on ice and in 4°C room.
Starter Culture Growth
Starter Culture Growth
Streak cell-stock on an LB agar plate, and grow o/n at 37 °C .
Inoculate a single colony from the plate into a 5 mL LB liquid culture and grow o/n at 37 °C with shaking.
Typically prepare TFB1/TFB2 solutions now, and store at 4 °C o/n before preparing cells in the next day.
Competent Cell Preparation
Competent Cell Preparation
Supplement 250 mL of LB in a 1 L flask with 20 millimolar (mM) MgSO4.
Inoculate with 2.5 mL of overnight 5 mL culture.
Add 5 mL of 1 Molarity (M) MgSO4 stock.
Grow cells by shaking at 37 °C until OD600 = 0.4 –0.6, typically takes02:00:00 – 03:30:00 for most cell lines.
| Cell Line | Inoculum | Estimated Growth Time | |
| Top10 | 2.5 ml | 2 h 45 min | |
| Whelan DH5ɑ | 2.5 ml | 2 h 30 min | |
| RIL | 2.5 ml | 4 h | |
| Stbl3 | 3.5 ml | 2 h 45 min |
Pre-chill buffers TFB1 and TFB2 on ice. Pre-chill p1000 tips at 4 °C .
Gently pellet culture by centrifugation at 4500 x g for 00:05:00 , 4 °C .
Note
4,500 x g = ~5,500 RPM in SLA-1500, SLA-3000 or F10BCI-FibreLite “F500”
Gently re-suspend the cell pellet in 100 mL ice-cold TFB1 (0.4 original volume).
Note
Essential to obtain optimal competency: complete the following steps on ice and while working at 4°C.
Pour off media, and place cell pellet on ice. Then gently re-suspend pellet while working on ice in 4 °C room.
Note
It is normal for the pellet to be somewhat difficult to re-suspend in TFB1 (should easily re-suspend in TFB2).
Incubate the re-suspended cells on ice for 00:05:00 in 4 °C room.
Gently pellet culture by repeating centrifugation at 4500 x g for 00:05:00 ,4 °C .
Gently re-suspend cells in 10 mL of ice-cold TFB2 (1/25th original volume).
Incubate the re-suspended cells on ice for 00:20:00 in 4 °C room.
Label and pre-chill 1.5 mL microfuge tubes on ice and in 4 °C room.
Aliquot cells as 220 µL stocks in 1.5 mL microfuge tubes on ice.
Flash-freeze aliquoted tubes by submerging in liquid-nitrogen, and store at -80 °C .
Note
Typically use 25–50 µl of cell stock for each transformation.
Tubes can be freeze-thawed multiple times with minimal competency loss.
Testing Competency of Cell Stocks (optional, recommended if high competency is required)
Testing Competency of Cell Stocks (optional, recommended if high competency is required)
Measure competency by transforming 0.1 ng of plasmid DNA (within 10 µL total dH2O volume).
Note
Optionally, also plate 50 µL of 1/10 dilution of transformation (in LB) mixture for easier colony counting.
Transform 10 µL of a 0.01 ng/µL stock of pGEM9z into 50 µL of competent cells and plate 50 µL on Amp+
LB plate.
Count number of colonies and calculate cell competency
Expected result
EX. Counted 832 colonies from 50 µl plate.
a. 0.1 ng of DNA in 0.560 ml = 0.17857 ng DNA / mL.
b. 0.17857 ng DNA / ml x 0.05 ml plated = 0.00892857 ng DNA.
c. 832 cfu / 0.00892857 ng = 9.318 x 104 cfu/ng → 9.32 x 107 cfu/µg.
Note
Optional: also plate cells on Amp+ plates to verify no background resistance.