Trick in competent cell - make me crazy (Mar/17/2007 )
Hi all,
By following the normal protocol, completent cell (E coli) were prepared.....but sometime I found that the completence is too high, but some time is so poor that I have to discrad the whole batch competent cell........
is there any trick that help to raise the competence ??
Indeed, because my insert were blunt end ligated into my vector, with the addition of low competence of those cell, no colony can form......... 
this is my protocol........should I modified it ???? THX all~~~ 
1. inoculate 1ml of overnight culture Top10/XL1Blue/ into 100ml LB medium and incubate at 37oC untill OD = 0.3-0.4
2. collect cells by centrifugation and remove the supernatant
3. resuspend in 2.5ml 100mM CaCl2
4. Store at or 4oC overnight
5. centrifuge and remove the supernatant, resuspend in 0.5ml 100mM CaCl2 with 15% glycerol
6. Aliquot, freeze it by liquid nitrogen and then store at -80oC
I use another method instead of CaCl2.
Just sharing.
If you store the resuspended cells at 4Celcius overnight, arent you killing the cells?
Cant you do it straight away by freezing in liq nitrogen?
Oh.. plus. blunt end ligated product is pretty hard to be transformed inside. 
Hi all,
By following the normal protocol, completent cell (E coli) were prepared.....but sometime I found that the completence is too high, but some time is so poor that I have to discrad the whole batch competent cell........
is there any trick that help to raise the competence ??
yes there is a way, you can add DnD solution , to prepare more number of competent cells
you must use freshely prepared hostcells.
it might depend of the starting colony. Some colonies grow to become good cells and others not so good.
Then you might have to go back a older stock and start by plating it again and picking new colonies.
I've used the One-step TSS protocol in Ausable's "Short protocols in molecular biology" book with great success. This super easy and efficient method was developed and described in a paper by Chung et al in PNAS a while back (attached).
Here's my short take on it: First make 2x TSS: 40% w/v PEG-8000 flakes, 10% v/vDMSO and 50mM MgCl2 in 2x LB; NB:it will take a little heat(~50`C) and time(~1hr) to dissolve the PEG. Add a little HCl to bring pH down to 6.5, and sterilize; this can be stored at room temp for a month or so, longer if you store at 4C, but the PEG precipitates, so you'll have to warm it to dissolve before use. Once you have grown an overnight culture, innoculate 1 ml of the O/N culture into 100 ml enriched LB (Millers LB+20mM d-glucose) or TB and grow to OD600 0.3-0.4- with JM109 and DH5a it takes about 2-hours. Place 3ng plasmid(<5ul) into a 5ml sterile polystyrene tube and hold on on ice. Add 100ul 2xTSS to tube and hold on ice for 10 min. Add 100ul of cell suspension, mix by vortexing and return to ice for up to an hour. This was the transformation step - no heat shock needed!- and you can store your transformed cells in this solution at -80`C for at least a year!!!! Next, add 1 ml pre-warmed SOC media to transformation solution, mix by pipetting a few times and incubate cells with shaking at 37`C for an hour to recover. Spread onto appropriate selective media plates and count colonies the next morning.
I use this method frequently, and have had only one failed transformation I can recall. Hope this helps. Cheers!-JAH
I didnt know TSS method is widely used as well. I used the TSS method too. So far, the transformation works for me.
To make the TSS buffer, there are several ways of doing it. Some did mentioned to bring the pH down to 6.5. Some didnt.
Well, my first time doing it, I didn't add any HCl to bring down the pH. But it didnt really affect my transformation. So is it really necessary?
Hehe
Thank you very much, all of you are very helpful
timjim, I just find that store it at 4oC overnight do not kill them, for the ideal case, the competence is so good that even blunt end ligation product can give 50 colonies......but well, sometime are useless.........
by the way, what is TSS stand for ???
TSS stands for Transformation Storage and Solution.
50 Colonies considered pretty low though.
Thanks for the clarification though.
Good morning to those in North America,
good afternoon to those in Europe,
what if I use 2.5 times as much LB for making TSS? Can I still use my TSS solution?
Thanks! Have fun
Catarina