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I would like to test if this is caspase mediated apoptosis, using the pan-caspase inhibitor Z-VAD-FMK. Does anyone know how long this peptide lasts in culture, i.e. how often should I add it over a 5-10 day period?
Can't seem to find literature on this, but maybe someone here knows? Thanks!
Just to make sure that You use low salt LB for the LB-Agar plates preparation.
Following transformation, the bacteria shoud be grown on low salt LB-Agar with Zeocin.
Yes. I do......have you worked with this plasmid?
Anyway, try these things if you haven't already: Linearize your vector with an overnight digest, gel purify both the vector and the insert after cutting, and heat inactivate the phosphatase. Replace your ligase buffer (it contains ATP, which goes bad after many thaws) and probably your phosphatase too (doesn't seem to be working very well).
The linerization worked well....I purified the vector and also the inserts from a gel. After gel elution I desphosphorylated (only vector ) and also deactivated at 65 degress for 20 min (its a brand new phosphatase - should work fine). Also ligase and ligase buffer are new...ligase buffer has just been thawed once, when I took aliquots which I then thaw when needed.
You see why I don't know what to do....it seems to me that I have already done quite alot....
But many thanks for the suggestions and please let me know if you have any other ideas!!
Ah, you do have a sticky problem.
This solution might seem a little strange, but empirically it worked when I had a similar problem. After you gel purify the insert, concentrate it by ethanol precipitation and bring it up in 10 uL TE. (I am assuming you use a kit to gel purify). Don't ask me why concentrating a Qiagen kit eluate of 30 uL to 10 uL by ethanol precipitation would make much difference, but it really did for me. (Perhaps it's an issue with salts being eluted with the DNA ... I don't know.)
Stick with the higher insert:vector ratios for the ligation (I usually calculate the ratio based on moles rather than mass; I would try 10:1 5:1 and 2:1 molar ratios). You might end up getting a lot of clones with more than one insert, but at least that's headed in the right direction.
Also, screen lots and lots of clones, if you have them. With any cloning project I do now that doesn't work right away, I screen 48 clones at a time, as long as I get that many (48 because benchtop microcentrifuges hold 24 miniprep columns, and screening 2 rounds of 24 minipreps seems to fit in one day of work). One time, I got a single clone out of 48 that was exactly right. And it only takes one...
It's not very elegant, but sometimes brute force will do it.
I have been reading the topics in this forum now for a while and discovered the expertise and wisdom that is here. You are the ones I need to ask.
I have a problem.
I have been struggling now already for quite some time with one lousy transformation (of my bait plasmid). And actually this is supposed to be only the first step of my Y2H screen I'm actually supposed to do (and I only have time until the end of this year since I'm doing my master thesis)...
This is the case: I have an 1,5 kb insert (Arabidopsis protein phosphatase 2A subunit B ) which has already been subcloned into pDrive and succesfully cut out of there with XhoI (I'm using single digestion). Now I need to clone it into the 4,8kb vector pHybLex/Zeo (Invitrogen).
This I have nicely linearised and desphophorylated with the anarctic phosphatase by NEB (no colonies on religation control plate).
I have tried the following ligation reactions and conditions:
- 10ng vector/100ng insert --> 4degrees celcius o/n,
- 10ng vector/60ng insert --> 4 degrees o/n,
- 100ng v/50ng i --> 4 degrees o/n,
All of these ligations resulted some colonies after transformation into Dh5alpha E.coli. But each time they were not containing an insert (mini test-digested with XhoI).
I also tried some other ligations conditions like 16 degrees and RT over night...they did not bring any colonies.
We are using T4-ligase from NEB.
I actually don't understand what is going wrong. Since the vector is dephosphorylated I would expect no colonies to appear if the insert is not in the vector. Still always some negative colonies appear.
I would appreciate every kind of help you can give me...what can I do differently? Does somebody have experience with the pHybLex/Zeo plasmid?
Not sure why your re-ligation negative control plate had no colonies; if you're getting empty vector colonies otherwise, it clearly should have.
Anyway, try these things if you haven't already: Linearize your vector with an overnight digest, gel purify both the vector and the insert after cutting, and heat inactivate the phosphatase. Replace your ligase buffer (it contains ATP, which goes bad after many thaws) and probably your phosphatase too (doesn't seem to be working very well). I always ligate @16 C o/n.
And don't lose hope!