Yeast transformation - low efficiency (Dec/15/2008 )
I transform the yeast using LiOAc/PEG method. The efficiency was low, about 10^3-10^4 colonies /ug plasmid.
The protocol was:
240 ul 50% w/v PEG3350
35 ul 1M LiOAc
25 ul carrier ssDNA
1 ul plasmid
Mix well and add to the washed cells.
30 degree C, 30 minutes
42 degree C, 20 minutes
spin down, resuspend in water
Is there anything wrong?
what kind of yeast are you working with?
If this is S.pombe, I would say you should increase the duration of the 30 C incubation to 1hr, reduce the heat shock (42 C) to around 10-5min (I personally use 5min) and add 42ul DMSO to improve the heatshock.
Also how hard are you spinning your cells? Spin for the shortest time possible at the lowest speed setting your machine can go. The yeast cells are fragile after the transformation protocol.
If you do add the DMSO, wash it off before plating the cells.
Once your cells are plated, keep your incubator humidified. S.pombe does not like dried plate surfaces. This helps improve growth rate and to a lesser extent recovery rate. So if you do use humidity and are colony counting make sure you do this for all your experiments.
Could you tell me abit more about what you do once your cell culture is ready. Do you wash in your cells in TE+LiAc before making a final DNA+cell mix?
Lastly the LiAc transformation protocol is a bit crude and can perhaps be pushed to around 10^5 colonies/ug DNA but not much more. There are better protocols, but are more complex and time consuming.
Thank you, perneseblue,
I am working with baker's yeast. The cells were pelleted by cfg at 3000 rpm, 5 minutes. Washed once with 1x volume water. The transformation mix( plamsid, ssDNA, PEG3350, LiOAc) was then added to the cell pellet. Mixed by vortexing for 10 seconds. After 30 minutes 30 degree C, and 20 minutes 42 degree C incubation, the cells were pelleted, resuspended in water, and plated on selection plates.
I will try to add DMSO next time.