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S.Cereviae electroporation:Chapter 2

bruno COLLINET bruno at epcm.u-psud.fr
Tue Jul 30 10:20:11 EST 1996


Few weeks ago I was asking for an efficient S.Cerevisiae electroporation 
protocol.
First of all, I would like to thank all the people who gave me many 
helpful advices.
Here are the answers they sent me :

	1°) from Abhijit Datta 
(Abhijit_Datta at macmailgw.dfci.harvard.edu)

there are some protocols available in the biorad catalogue that comes 
with the biorad electroporator, but I found the following to work 
OK...for maximal efficiency use the published protocols...

1) Harvest exponentially growing cells from 50ml culture (approx. 
1X10e6). 
Wash 3 times with distilled water (chilled).  Wash 2X with 10% glycerol
(chilled).   Resuspend in 50-100ul vol.  

2) Make clean DNA by precipitating and washing 3X. OR use Qiagen preps. 
Dilute DNA 10-100 fold (compared to LiAC protocol usage) in 10% 
glycerol.  Add
5ul max DNA vol to 40ul cells in a chilled 0.2mm disposable cuvette.  
Set
controls at 1.5Volts pulse, 25 for capacitance and 200 for resistance.  
(the
last 2 same for coli transform using same cuvettes).  

3) Wash out cuvette with 500ul cold 10% glycerol immediately and plate
immediately.



	2°) from Mark Walberg  (walberg at simmons.swmed.edu)

Subject:Yeast EP

Hi,  Here is an easy protocol that I put together for electroporation of
yeast.  You can just scrape the colonies from a plate, wash the cells, 
add DNA and zap.  The efficiency is a little higher for cells grown in 
liquid, though.
 Good luck,    Mark Walberg

A QUICK METHOD OF TRANSFORMING YEAST BY ELECTROPORATION
M.Walberg   2/3/91

This has worked with a few different strains. There is variation in 
efficiency between strains. It works well with miniprep circular DNA as 
well as with lower efficiency events such as integration and allele 
rescue by gap repair of a linear DNA. So far, none of the parameters 
have been optimized, so there is likely to be room for improvement.

1)	Scrape enough yeast cells from a plate to provide at least a 
20-40 ul volume of cells for each transformation. Put cells into cold 1M 
sorbitol in a microfuge tube and vortex  vigorously to disrupt clumps. 
The water used to make the sorbitol solution must be low in ionic 
strength.

2)	Spin at low speed (e.g. about 3000 rpm) in a microfuge for long 
enough to pellet cells. (2 minutes at this speed works well.) Discard 
sup.

3)	Resuspend cells in about 1ml of cold 1M sorbitol and repellet. 
Repeat this for a total of three washes. If you are doing a large volume 
of cells, then you might want to do more washing to be sure to get rid 
of enough of the conducting solutes.

4)	Suspend washed cell pellet in up to an equal volume of 1M 
sorbitol. I have not tested this yet, but I think that transformation 
efficiency increases with increasing cell density.

5)	Mix 20-40 ul of cells with not more than about 1/10 volume of 
DNA in a low salt buffer (e.g. TE or TE/5). Then, put the cell/DNA 
mixture into a cold cuvette and electroporate. I've used the 1mm 
cuvettes with settings of 1.5 kv, 25 uF, 400 ohms (using a BioRad 
Genepulser). This gives time constants between 5 and 8. (The maximal 
time constant at these settings would be 10.) 200 ohms works fine, too, 
giving time constants half of those obtained at 400 ohms. I haven't 
tried other cuvettes or settings with this method yet. The cuvettes can 
be reused many times, by washing a few minutes in bleach, rinsing very 
well with water, and then resterilizing in ethanol for a few minutes.

6)	Quickly after electroporating, wash cells out of cuvette with 
cold 1M sorbitol (I generally use about 200-500 ul) and spread on a sorb 
plate using the desired selection. Colonies are visible in 1 1/2-2 days 
with the strains I've used.

Efficiencies probably are roughly 104/ug of circular CEN plasmid DNA or
better. Miniprep DNA works well, with 1/30th of a miniprep (of pUC based
plasmids) yielding a few hundred colonies. Transforming with linear DNA 
(e.g. for integration or allele rescue) is less efficient, as expected, 
but so far has worked every time. The efficiencies noted above are 
obtained using cells grown in rich medium. 
If cells are grown on minimal medium (e.g. to select for a marker) the
efficiencies tend to be much lower.  So, if at all possible, grow the 
cells in a rich medium.
A slightly higher efficiency can sometimes be obtained by growing the 
cells in suspension instead of on plates as in the Becker and Guarente 
protocol in Meth. Enz. 152.  The only change that I make in this 
protocol is that I do 3-4 washes in 1M sorbitol in 50 ml. Falcon tubes 
instead of the large volume water washes described in the original 
protocol. The smaller volume washes lead to less loss of cells during 
the washes.



	3°) from Pierre Falson (falson at verlaine.saclay.cea.fr)

We are currently use electroporation ofr yeast without any problem.
here follow these conditions:
taking a 3 weeks-less-old colony, growth in 100 ml Yeast Extract 1%,
bactopeptone 1% and glucose 1%. Go to 1.3-1.5OD600nm/ml (10E8cell/ml).
Centrifuge in two falcon tubes two times at 5000 rpm and wash with 100
ml of sterile, cold water, centrifuge, wash with 50 ml water,
centrifuge, suspend in 4 ml 1 M sorbitol (sterile, cold), centrifuge,
suspend in 0.1 ml of 1M sorbitol (s, c). let the yeasts at 4°C. they can
be used immediadely or one or two days after. yeasts are aliquoted in 40
µl fraction, mixed with 0.1µgDNA/0.1-5µl buffer (TE). the mix is let on
ice for 4 min. the apparatus (biorad) is setted to 1.5kV, 25 µF (gene
pulser) and 200 Ohms (pulse controler). pulse in a 2 mm chamber (time
constant is 4.5 - 5 msec). add 1 ml of 1 M cold sterile sorbitol, pour
on plate. colonies will appear in 48-72 h. I garanty this protocol!
Pierre Falson


	4°) from Christian Velten (cve at ifm.mh-hannover.de)

NB:Christian Velten uses the same protocol as Pierre Falson with the 
improvments below.

> we are currently use electroporation ofr yeast without any problem.
> here follow these conditions:
> taking a 3 weeks-less-old colony, growth in 100 ml Yeast Extract 1%,
> bactopeptone 1% and glucose 1%. Go to 1.3-1.5OD600nm/ml (10E8cell/ml).
> Centrifuge in two falcon tubes two times at 5000 rpm and

You can increase the transformation rate by putting the cells after 
first
collection for 45 min into LiAc/TE (20 ml/100 ml culture) and adding DTT
(500 microl 1M DTT/20 ml) for another 15 min. Then ...

> wash with 100 ml of sterile, cold water, centrifuge, wash with 50 ml 
>water,
> centrifuge, suspend in 4 ml 1 M sorbitol (sterile, cold), centrifuge,
> suspend in 0.1 ml of 1M sorbitol (s, c). let the yeasts at 4°C. >they can
> be used immediadely or one or two days after. yeasts are aliquoted in 
>40
> µl fraction, mixed with 0.1µgDNA/0.1-5µl buffer (TE). the mix is let 
>on
>ice for 4 min. the apparatus (biorad) is setted to 1.5kV, 25 µF (gene
> pulser) and 200 Ohms (pulse controler). pulse in a 2 mm chamber (time
> constant is 4.5 - 5 msec). add 1 ml of 1 M cold sterile sorbitol, pour
> on plate. colonies will appear in 48-72 h. I garanty this protocol!

You can also freeze the final Sorbitol-suspension. Max. decrease of
transformation rate about 10%.

Christian
"Two bands or not two bands? - THAT is the question!"


	5°) from Michael Lichten (lichten at helix.nih.gov)

1)Use a 5ml overnight grown in YPD.  Make it from a fresh colony.  Wash
the cells 3x in 5 ml water, 2x in 1M sorbitol, and resuspend the pellet 
of cells in whatever 1M sorbitol remains after pouring off the second 
wash.

2)We have found that the addition of carrier DNA (12.5
ugm/transformation) enhances the yield of integrative transformants 
about 10x. See Schiestl and Gietz (Curr Genet 1989 Dec;16(5-6):339-46) 
for how to make carrier.

3)  We find that plating at lower cell concentrations often helps 
recover transformants.  If you put too many cells on a single plate, 
they'll eat up all the goodies.
Good luck!

	6°) from Olaf Lange (Olaf.Lange at Schering.DE)

Subject: electroporation
here the electroporation protocol for yeast (s.cerevisiae):

reference: current protocols in molecular biology, supplement 18, 
13.7.5-7

1. preculture
inocculate 1oml YPD medium with an single colonie aof your yeast strain 
and let it grow over night at 30 deg. C with shaking

2.culture
inocculate 50ml YPD medium with 0.5-1ml of your preculture and let it 
grow at 30 deg.C until OD600=1.3-1.5 (generationtime is about 90 min).
this may can take a lot of time so that I inocculate the culture with 
smaler volumes than 0.5ml and let it grow over night.

3. harvest
centrifuge the cells for 10 min at 4 deg. C and 4000xg

4.wash
resuspend the cells in 8ml icecold water

5. increasing competence (optional)
add 1ml 10xTE pH 7.5 , 1ml 1M Li-acetate; mix carefully and incubate at 
30 deg C and 90rpm for 45min add 0.25 ml 1M DTT, mix carefully and 
incubate for further 15min under same conditions

6. concebtrating cells
add 40ml icecold water, centrifuge for 10min at 4000xg and 4 deg. C.
wash the cells first with 25ml icecold water,centrifuge, then with 2,5ml 
1M icecold sorbitol,centrifuge, and at least resuspend cells in 50l 
icecold 1M sorbitol the whole volume aof the suspension should be  
100-150 l

7. electroporation
transfer 40 µl cell suspension in icecold 0.2cm electroporation cuevetts 
and add <100ng DNA with a volume smaller than 5 µl.
leave the cuevetts on ice do not use carrier-DNA
puls :  1.0 kv
        25  F
        600  
        t=13-13.5 msec
after puls add immediatley 1ml icecold 1M sorbitol and transfer the 
suspension to prechild 1.5 eppis. leave the eppis on ice till plating
plate 100, 200 and 700 µl on slective agarplates



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