Problem with transformation using electroporator from eppendorf - (Jun/09/2006 )

Hello,

I would like to know anyone has worked with the electroporator of the eppendorf company.
I did the preparation of electrocompetent cell and the other steps as per their protocol but dint get the transformation.
Any suggestions on this topic kindly reply immediately.

hi - perhaps a bit more information might help us to help you better
- no transformation - what controls did you do? did you get any colonies at all? only empty vector (ie. is it your ligation)? correct antibiotic concentration? which strain of e.coli are you using?

hi

let me tell u the whole waht i did then possibly u will be able to tell me..
the strain of E.coli i used for transformation is DH5 alpha and i used pUC18 to transform the procedure i used to make electrocompetent cell is given in the attachment ...so i prepared the electrocompetent cells of E.coli as per protocol....
it was said in the protocol to take 1uL DNA of pUC (10pg of in water) to the 40uL of eletrocompetent cells i did and gave a 1700v for 5ms and plated this on LB plates having 50ug/mL of ampicillin and the X-gal and IPTG was spread plate and dried for 30 min before spread plate of the electroporated culture. I havent used any foreign DNA for ligation i have juz used pUC 18 to transform the E.coli DH5 Alpha. so i shud be getting blue colonies in this plate but there happen to be no colonies but when this electropoated sample was plated into LB without amp+X gal+IPTG there was mat formation of growth of E.coli this was kept as control. so waht will u say about this?
I think there was no transformation of the E.coli with pUC 18 using the electroporator.
Iam repeating again and have increased the washing step to 5 for making electrocompetent cells this time and have got very few colonies on only ampicillin plate with no xgal and iptg and have increased the DNA conc of pUC 18. i added 5uL of pUC 18 to 100uL of electrocompetent cell. the results are a few colonies. can u tell what could be the problem???

How certain are you of the quantity of pUC19 in the sample? How was it prepared? A common problem with electroporation is arcing of the sample due to high salt concentration in either the cells or the DNA added to the cells. Did the electroporator make a loud POP when you activated it? If so, then this is your problem. You would need to wash your cells more effectively and purify salts out of your DNA sample.

i agree with phage....also, are you using the appropriate cuvettes for the electroporation - sometimes easy to confuse with the yeast ones???

Iam using the 100uL capacity cuvettes. and tell u of the preparation of plasmid. the pUC 18 Concentration of stock was 250ug/mL from which a working stock was preapred taking 1uL of vector and made up with 999uL milliq. from this working stock i had made a 4uL+96uL to get 10pg in water so as to use it for instant uses. according to the protocol they said 1uL (10pg in water) is added to 40ul electrocompetent cells and so i did...no results in LB+amp plates....think the concentration is low when compared to the cell /mL. more info please pass on.

The first thing I would try is to transform much higher concentrations of pUC19, until I obtained transformants. You might also want to verify that the pUC19 sample you have is correct by chemically transforming some cells with it. Plate it out on the same plates you use for electroporation, and make sure the plates/antibiotic are not a problem. When this is working, reduce the concentrations and figure out what is going on. You could also run a gel of your pUC19 sample. Sometimes the DNA concentrations are wildly off due to genomic DNA contamination, phenol contamination, or instrument error. If you look at how bright the band is compared to a DNA ladder, it will be obvious if this is a problem.

are you sure your e.competent cells are working? i mean, are they alive? and did you check the transformation efficiency of your cells? they may have low efficiency. u may transform a small vector like pKS which is easy to transform in order to see how efficient is the transformation with e. competent DH5alpha cells you prepared.
i always check mine with pKS..