transformation step: 5min or 30min? - (Jan/25/2006 )

For doing e. coli transformations I have been following the protocol given to me by someone in the lab. I have concerns about the third step:

1) thaw competent cells on ice
2) add dna
3) leave on ice for 5 minutes
4) heat shock at 42degress for 30-45 seconds
5) ...


From the protocols I have found on the web, the third step is always 30 minutes, not 5.

I've been having difficulties with transformations and was wondering if this could be (part of) the cause.

Would there be a big difference between 5 and 30 minutes? If so, why? I'm using DH5 and BL21 strains.

I use 3 mins with good results

I'm old school -- I always go 40 minutes on ice, heat shock 45 seconds at 42°C. I suspect 5 minutes is adequate, but I've been doing it this way for 15 years, and I'm kinda set in my ways...

im incubating 1 min acc to molecular cloning.... ....

I do thirty minutes.

I've also done it for five minutes...never really noticed a difference.

I go 30 min ice,
heat shock 42 degrees for thirty secs,
ice for 1 min,
add room temp SOC (5xvolume of cells)
Incubate for at least one hour (longer is better), shaking at 37 degrees celsius,
plate on antibiotic media.

-Matt

Maybe you're trying to transform too much DNA.
In transformation "less is more"!!!! So I never transform more than half of my ligation or in the case of plasmid-transformation 10 picogram DNA.
Of course your cells have to be of good quality. So if you have troubles with transforation you should test your competent cells by transforming 10pg of plasmid DNA.

- I thaw the competent cells on ice for 10 min
- I add my DNA and let it sit on ice for 10min.
- instead of 42C I put eveything at 37C for 5 min.
- add SOC and incubate on shaker at 37C for 45 min to 1 hour.
This always works for me.

Anouk

anouk has a good point -- if you're transforming your cells with ligation mix, too much is a bad thing. I usually go no more than 2 ul of ligation mix per 50 ul of cells...

How do you do your ligation? How are your competent cells prepared?

For the ligation, I do a rapid ligation in 10 minutes using max 200ng of DNA with a 3fold molar excess of insert. The total ligation volume is 20ul, so there is approx 10ng/ul.

I had been using 20ng per transformation (200ul of cells); yesterday I tried 10ng and waited 30min after adding the ligation mix and got 0 colonies on 4 plates - plated 1, 10, 100 and 200ul.

I don't know how the competent cells were prepared. I'll have to ask.

I should say, I'm a computational biologist who's new to getting my hands dirty and tend to just do what others in my lab do as to protocol. I was wondering about SOC. I was told to just add LB. Could this be making a big difference?

I'll give some more details regarding the vector I'm trying to create:

1) digestion of the plasmid with EcoRV and SacII (heat inactivation of enzymes)
2) SAP treatment of plasmid
3) T4 treatment of insert (the insert is 25bp in length and was made by annealing oligos. One end is blunt, for EcoRV site, and the other has an overhang for SacII)
4) heat inactivation of SAP and T4 kinase
4) ligation

Apart from the transformation, the only other step I don't feel confident about is the digestion. From what I understand, SacII has very poor efficiency.

you mean you are not using SOC after shock? but you must to I think....that is super enriched medium for your cells to survive after shock....

After heat shocking cells, you should afterwards grow them at least 1 hour @ 37 and shaking in SOC medium for them to recover from the shock. (LB instead of SOC works too I'm told but less bacteria recover).
SOC is indeed super rich medium, specially made for cells to recover from heat shock.