Protocol Online logo
Top : Forum Archives: : Molecular Biology

Introduce BAC DNA via heat-shock - (Oct/04/2006 )

Hello, everyone,

This is the first time i use the forum. I know people routinely use electroporesis to introduce BAC DNA, but since we do not have an electroporator in our lab (I can borrow one from other labs though), I am thinking if it's possible to transform cells via heat-shock? Please advise.

Thank you! cool.gif

-Biomedmedia-

yeah, chemical transformation. It is possible. But the efficiency is not good, becomes worse as the BAC grows bigger. I would beg and borrow an electroporater. It would save lots of heartache.

-perneseblue-

QUOTE (perneseblue @ Oct 5 2006, 12:46 AM)
yeah, chemical transformation. It is possible. But the efficiency is not good, becomes worse as the BAC grows bigger. I would beg and borrow an electroporater. It would save lots of heartache.


Thank you! perneseblue!

By the way, what are the factors determine the time constant? Generally it should be around 5 milliseconds, but i don't know why this time, i got only 2-3 milliseconds. What's wrong? Please help!!!

Thanks!

-Biomedmedia-

time constant

t = RC

where R = resistence and C is capacitance.

Since the capacitance is set by the electroporater user (For E coli is usually 25uF)
Thus the main variation in time constant is caused by the resistence of the (cell + DNA) mix in the electroporation cuvatte.

Both the presence of salt and/or "high" concentrations DNA in the electroporation mix will decrease the mixture's resistece and thus time constant.

Salt can leak in from either the DNA that is added or via the competent cells, more so if the cells are home made). And if the cuvettes are recycled, improper cleaning can also cause salts to come in.

-perneseblue-

QUOTE (perneseblue @ Oct 6 2006, 02:08 AM)
time constant

t = RC

where R = resistence and C is capacitance.

Since the capacitance is set by the electroporater user (For E coli is usually 25uF)
Thus the main variation in time constant is caused by the resistence of the (cell + DNA) mix in the electroporation cuvatte.

Both the presence of salt and/or "high" concentrations DNA in the electroporation mix will decrease the mixture's resistece and thus time constant.

Salt can leak in from either the DNA that is added or via the competent cells, more so if the cells are home made). And if the cuvettes are recycled, improper cleaning can also cause salts to come in.


Thanks again, perneseblue! For yesterday's experiment, I used SW102 cells (modified E. Coli from NCI-Frederick for recombineering) directly from glycerol stock (40% final concentration of glycerol). I didn't specifically prepare the bacteria in the regular way people make electrocompetent cells (by washing sequentially with 10% glycerol) because they are electrocompetent cells already. I used 0.2 cm cuvette (that's the only one i can find in the lab), with capacitance of 25uF, and i got ~3.5 millisecond time constant and an arcing, w/o beeping...

Since you said the resistance depends on cell+DNA mixture, I am wondering if the cell density is too low (it has not been concentrated, instead, diluted half with 80% glycerol). will this explain?

Thanks again!

-Biomedmedia-

I am quite concern of the arcing that you have observed. Arcing in a cuvette means that the cells are being electrocuted! Ie the cells have been fried and are now quite dead.

Is it certain that the SW102 cells from NCI-Frederick are electrocompetent, rather then chemical competent? (Or for that matter, are the cells even in a state of conpetency?) The wording of "40% glycerol stocks" strikes me more as a "frozen storage". I could just be paranoid.

But in anycase, I don't think, bacteria concentration plays a major role increasing resistence. If anything, I would bet high bacteria concentration decreases electrical resistence (shortening the time constant).

And by the way, what voltage do you use? I looked up my lab's cuvette guide, 0.2cm gap (for bacteria) uses 2.5kV. Could the setting for the voltage be too high and the cause of the sparking (and also the reduced time constant?)

-perneseblue-

You need to prepare the cells for electroporation. Grow them to OD 0.5, chill on ice, and wash 3x with ice cold DI water with a 4C centrifuge. Resuspend in 1/10th volume ice cold DI water, keep on ice, and use for electroporation. You can't electroporate cells directly from frozen glycerol stocks. They are too conductive, and they are not at high enough density. See the protocol section on preparing electrocompetent cells.
If you get arcing, then you should dialyze your DNA against DI water with the floating membrane method.

-phage434-

QUOTE (phage434 @ Oct 6 2006, 09:43 PM)
You need to prepare the cells for electroporation. Grow them to OD 0.5, chill on ice, and wash 3x with ice cold DI water with a 4C centrifuge. Resuspend in 1/10th volume ice cold DI water, keep on ice, and use for electroporation. You can't electroporate cells directly from frozen glycerol stocks. They are too conductive, and they are not at high enough density. See the protocol section on preparing electrocompetent cells.
If you get arcing, then you should dialyze your DNA against DI water with the floating membrane method.


Again, thanks perneseblue and phage434!

@perneseblue, i used 1.75V, the voltage is not that high, but as phage434 said, i should probably prepare electrocompetent cells as he said. It turns out NCI-Frederick said so.

@phage434, i found the protocol for preparing electrocompetent cells. In case other people need it, i post the link here:
http://www.its.caltech.edu/%7Ebjorker/Prep...ocomp_cells.pdf

Great help from you guys! Thanks alot and have a wonderful weekend!

-Biomedmedia-