TITLE: Preparing Chemically Competent E coli for Transformation
AUTHORS: Stephen Floor

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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). </div><div class = "text-block"><span>Adapted from Promega Cloning Manual Notes and Inoue/Hanahan protocol, DNA Cloning 1985. With help from Amy Lee, Brandeis (</span><a href="http://www.bio.brandeis.edu/leelab/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">http://www.bio.brandeis.edu/leelab/</span></a><span>). </span></div></div>

[STEPS]
?. [Starter Culture Growth]
Streak cell-stock on an LB agar plate, and grow o/n at .
37 °C
?. [Starter Culture Growth]
Inoculate a single colony from the plate into a  LB liquid culture and grow o/n at  with shaking.
5 ml
37 °C
?. [Starter Culture Growth]
Typically prepare TFB1/TFB2 solutions now, and store at  o/n before preparing cells in the next day.
4 °C
?. [Competent Cell Preparation]
Supplement   of LB in a 1 L flask with  MgSO4.
250 ml
?. [Competent Cell Preparation]
Grow cells by shaking at  until OD600 = 0.4 –0.6, typically takes –  for most cell lines.  ABC1Cell LineInoculumEstimated Growth Time2Top102.5 ml2 h 45 min3Whelan DH5ɑ2.5 ml2 h 30 min4RIL2.5 ml4 h5Stbl33.5 ml2 h 45 min
37 °C
ABC1Cell LineInoculumEstimated Growth Time2Top102.5 ml2 h 45 min3Whelan DH5ɑ2.5 ml2 h 30 min4RIL2.5 ml4 h5Stbl33.5 ml2 h 45 min
?. [Competent Cell Preparation]
Gently pellet culture by centrifugation at  for  , .
4 °C
4,500 x g = ~5,500 RPM in SLA-1500, SLA-3000 or F10BCI-FibreLite “F500”
?. [Competent Cell Preparation]
Gently re-suspend the cell pellet in  ice-cold TFB1 (0.4 original volume).
100 ml
Essential to obtain optimal competency: complete the following steps on ice and while working at 4°C.
?. [Competent Cell Preparation]
Incubate the re-suspended cells on ice for  in  room.
4 °C
?. [Competent Cell Preparation]
Gently pellet culture by repeating centrifugation at  for ,.
4 °C
?. [Competent Cell Preparation]
Gently re-suspend cells in  of ice-cold TFB2 (1/25th original volume).
10 ml
?. [Competent Cell Preparation]
Incubate the re-suspended cells on ice for  in  room.
4 °C
?. [Competent Cell Preparation]
Aliquot cells as  stocks in  microfuge tubes on ice.
220 µl
1.5 ml
?. [Competent Cell Preparation]
Flash-freeze aliquoted tubes by submerging in liquid-nitrogen, and store at .
-80 °C
Typically use 25–50 µl of cell stock for each transformation.Tubes can be freeze-thawed multiple times with minimal competency loss.
?. [Competent Cell Preparation]
Pre-chill buffers TFB1 and TFB2 on ice. Pre-chill p1000 tips at .
4 °C
?. [Competent Cell Preparation]
Label and pre-chill  microfuge tubes on ice and in  room.
1.5 ml
4 °C
?. [Testing Competency of Cell Stocks (optional, recommended if high competency is required)]
Measure competency by transforming  of plasmid DNA (within  total dH2O volume).
0.1 ng
10 µl
Optionally, also plate   of 1/10 dilution of transformation (in LB) mixture for easier colony counting.
?. [Testing Competency of Cell Stocks (optional, recommended if high competency is required)]
Count number of colonies and calculate cell competency
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.
Optional: also plate cells on Amp+ plates to verify no background resistance.
?. [Competent Cell Preparation]
Inoculate with  of overnight  culture.
2.5 ml
5 ml
?. [Competent Cell Preparation]
Add  of  MgSO4 stock.
5 ml
?. [Competent Cell Preparation]
Pour off media, and place cell pellet on ice. Then gently re-suspend pellet while working on ice in  room.
4 °C
It is normal for the pellet to be somewhat difficult to re-suspend in TFB1 (should easily re-suspend in TFB2).
?. [Testing Competency of Cell Stocks (optional, recommended if high competency is required)]
Transform  of a 0.01 ng/µL stock of pGEM9z into  of competent cells and plate  on Amp+          LB plate.
10 µl
50 µl
50 µl