Yeast Competent Cells - (Feb/06/2008 )

People were saying that doing sequential transformation would give higher transformation frequencies. However, these folks were preparing their cDNA libraries in bacteria so they had LOTS of prey to work with.

Anyways, I called Clontech and they said that doing the co-transformation (as per the kit instructions) give highest transformation frequencies. So I'm going to go with their recommendations. Hopefully I'll perform the screening this weekend.

Hi Kelly,

We use a method to amplify our cDNA further after the purification down the column, this way we should be able to conserve the reagents (as they are so expensive),it basically involves diluting the cDNA you have generated and performing a 10 cycle PCR with the 5 and 3' primers followed by a 'primer chase'. This way we hope to be able to generate lots of cDNA. I can send you the method if you like ?

Good luck with your screen, im testing some bait at the moment, hopefully do a screen next week.

Steve

Hi Steve,

Yes, please send or post the protocol. Has someone in your group already tested the PCR amplified library and it works well?

I've yet again slipped into transformation problems. So frustrating! People I know who do two-hybrids were suggesting growing the 3ml starter culture to stationary phase (which is easier to schedule). I tried that and that doesn't work for Y187. So, now to get the 3ml culture to not be overgrown I have start it around midnight.... I did one last transformation efficiency control experiment and if it looks good tomorrow then onto screening.

Have you performed the screening yet? Good luck!

Kelly

Hey Kzinn,

I was supposed to send you the protocol for amplifying cDNA but forgot ! So sorry, here it is.

I use 0.2ml PCR tubes and KODXL polymerase.
Dilute purified cDNA 1/50.
Set up PCR in 20ul volume (the protocol I found specifically states to use this volume and not larger), dNTPs 0.2mM, cDNA Amplimer primers 0.1uM and 1ul of your diluted template.

95C 30sec

95C 10sec
72C 6min plus 5sec for each additional cycle i.e 2nd cycle 6min 5 sec, 3rd cycle 6min 10 sec etc. These two steps for ten cycles.

Pause just before the end of last 72C extension and do a 'primer chase', add more primers to a final conc of 0.2uM (I keep my primer stocks at 10uM). Then do the following cycles.

77C 1min
65C 1min
72C 3min

I then purify using Qiagen PCR clean up, this is not ideal in terms of size exclusion but the columns with the kit are horribly expensive. I have run out cDNA made according to the clonetech kit and cDNA made this way and I cant see any obvious differences in the sizes of species represented ( a little crude I know). I found I make between 0.5-0.8 ug per reaction.

I have done a screen but not detected any interactions yet, although I got lots of colonies but they may have come out of a lawn, so im not sure. At the moment im doing a positive control with some cDNA that we know interacts with some of the promotors im interested in.

How is your stuff ?

Hi Steve,

Thanks for posting the protocol. Those columns are brutally expensive, but it seems like your method keeps the products of similar size.

I did two screens. I did one and plated on 100mm plates and got a ton of background, also my bait didn't go in very well (only 200k clones) so I didn't screen enough colonies. I re-screened and plated on 150mm plates and things worked better. I screened 1.2 million clones and got about 32 "positives." Although that is pretty subjective because even on the larger plates there was an awful amount of background. I choose colonies that were visible at 3 days and then let them grow another day. If the colonies grew bigger I continued on with them. I've re-streaked the positives and I will see tomorrow if they can grow to single colonies on selection media. If I get anything to grow then I will do colony PCR and sequence the products so see if there's anything interesting. I sequenced a few of the colonies that grew on the first screen - I got a ribosomal gene

Hope you find something interesting! Good thing you have some other cDNA to test as positive interactors. I'm using a 18bp repeat that I found doing EMSA. So I have no idea what this protein might be since the binding site has no homology to known elements.

Good luck!
Kelly

Hey Kzinn ( or anybody else reading this ),

Have you had any problems doing PCR from yeast as I cannot get it to work.

I have tried,
1,Touching a tip to a colony
2,Resuspending a colony in water boiling for 5 min,vortex, spin down and using various amounts as template.
3,As number 2 but performed in 20mM NaOH.

I can PCR from a plasmid but when I put the same plasmid in to yeast I get nothing. Also when I purify the plasmid from the yeast I get no product but when I transform this plasmid (after its purificartion from yeast ) in to bacteria and extract plasmid I get my expected PCR product ?! I have tried temp gradient and increasing cycle number (tried between 25-35).I using a hotstart taq from promega, are there polymerases that work better with yeast extracts.

thanks in advance for any help.

Steve

Hi Steve,

I've had no problems with colony PCR, and I don't do anything special. I use GoTaq from Promega and just do a 20ul reaction. I get enough product for a gel and sequencing. I just touch the colony, too. I use T7 and the 3' cDNA amplification primer (that came with the kit) and those two amplify at 50C annealing temperature, use 5 min extensions (3 min I found to be too short) and 30 cycles.

I haven't isolated plasmids yet. Do you use a kit or do you have a protocol from your lab? I've been told that plasmid isolation from yeast isn't easy, so what you describe doesn't sound out of the ordinary. Your strategy of transferring to bacteria with subsequent sequencing sounds good. I have isolated plasmids from Agrobacterium rhizogenes and tumefaciens (which we use for plant transformations) and the plasmid that comes from those preps always have to be transferred to E.coli for further processes.

I restreaked the 30 postives I got and they all regrew. I've sequenced most of them and a few interesting things popped up. Now I just need to get those plasmids out (sounds like fun) and test a mutated bait.

Hope you get some good results!
Kelly

Hi,

Thanks for that info ive been trying this for what seems like forever. To get the yeast plasmid out I used a kit from G-Biosciences, its called the 'EZ Yeast Plasmid Miniprep' and you are able to do it straight from a healthy colony. The most important ingredient looks like the zymolase to break the cells down, I got a free sample of the kit so im sure you can do the same. I transformed 5ul of this in to standard DH5 alpha and got colonies back from this (although it wasnt very efficient but hey you only need one right !

When you do the colony PCR does it work if you take colonies from the fridge or do they have to be fresh ? ive read conflicting reports.

Thanks again for help and well done on getting something interesting, heres to hoping I can catch you up !

Steve

Hi Kelly,

Sorry just to clarify in my head you are using a normal 3 step PCR programme i.e 94C 30sec/50C 30sec/72C 5min and noit the 2 step in the manual.

sorry to bug you sop much,agaion cheers for the help.

Steve