Tip for transformation : give me your opinion please! - (Jun/14/2006 )
Hello,
I don't manage to obtain positive clone after my transformation, so Iwould like to try this tip, find in a toping dating of jan/26/2005.
Have you ever try? What do you thing of this tip? iF you have already tried it and it worked, what is your exact protocol?
Thank you.
Hi all,
i am digesting my vector with 2 diffrent rest enzymes(Bgl II + Mlu I) yeilding diff sticky ends. after digesting do i have to proceed to dephosphorylation or can i skip it. in theory the sticky ends generated above would prevent self-ligation right?
i want to know is dephosphorylation is absloutely essential in such cases.
thx
rajgene
-rajgene-
--------------------------------------------------------------------------------
In theory it's not absolutely necessary to dephosphorylate if you are using two REs with cohesive ends, but in fact, if the restriction efficiency of the enzymes is not 100%, you will get some molecules which have been cut only with one or the other enzyme, so they will religate if they are phosphorylated. One useful trick when cloning using two different sites is checking if you loose any site in the vector multiple cloning site (if any site is between the two REs you are using and not anywhere else in the vector). If this is the case, and this site is not found neither in your insert, you can digest your ligation with that enzyme and you will cut only your religated plasmids and not the ones with your insert, which will not have that site. So you will get only colonies with the insert.
-badcell-
hi
i've done that and an other protocol. Assuming a vector holds ABCD restriction sites
Considering first try. I use sites A and D for ex. My insert does not contain C. So after ligation i precipitate with butanol ethanol wash and do a 1h restriction digest using C enzyme. Then precipitate and electroporation classical way.
In second example, i use for cloning A and D but my insert holds B and C sites. So i first do a A/D digestion, purify, and then do a B/C digestion. Purify again by column, and proceed for ligation.
I have not done this but seems like a brilliant idea. Will try it out in the future.
I did it. Of course, it works. 
Reduces the background of colonies containing only religated vector.
You can achieve a similar result by doing a PCR reaction to fabricate the plasmid backbone. This produces a linear product which then is digested to produce the correct ends. The ends can be added to the PCR primer, giving control over the properties of the "cloning site" by choice of the primers. Again, near zero background.
Thank you for your answers!
I made my ligation yesterday (150 ng plasmid in 15 µl), and today, I will try to transform with this tip.
I'm a beginner in molecular biology.
So, to precipitate, my ligation reaction, I must had salt, 2 vol ethanol 100%, -80°C for 30 min, centrifugation, wash my pellet with ethanol 80%. And after you resuspend your pellet in EB or directly in the buffer of your restriction enzyme? Which volume?
It's a bad idea to purify with column?
u may not resuspend your pellet in buffer of your enzyme. buffers are generally 10x, so you will use 3 microliter for a 30 microliter restriction reaction for ex. you may resuspend the pellet in water 
I made my ligation yesterday (150 ng plasmid in 15 µl), and today, I will try to transform with this tip.
I'm a beginner in molecular biology.
So, to precipitate, my ligation reaction, I must had salt, 2 vol ethanol 100%, -80°C for 30 min, centrifugation, wash my pellet with ethanol 80%. And after you resuspend your pellet in EB or directly in the buffer of your restriction enzyme? Which volume?
It's a bad idea to purify with column?
Thank you very mucho!!!!
How can I loose that!! I’m trying to fill in a vector (with klenow) to loose the NotI site, I’m having problems because I have only religated (not filling) colonies. I’m going to digest with NotI before transformation to cut all the background!! 
Thank
i've done that and an other protocol. Assuming a vector holds ABCD restriction sites
Considering first try. I use sites A and D for ex. My insert does not contain C. So after ligation i precipitate with butanol ethanol wash and do a 1h restriction digest using C enzyme. Then precipitate and electroporation classical way.
In second example, i use for cloning A and D but my insert holds B and C sites. So i first do a A/D digestion, purify, and then do a B/C digestion. Purify again by column, and proceed for ligation.
Hi,
I am currently trying to ligate an insert into a vector using EcoRI and HindIII sites, but keep getting religated vector. I am very interested in this method of using a third restriction enzyme (PstI).
I am a bit confused by the protocol though. SO, firstly I do my usual digestion of the vector (HindIII and EcoRI), which removes the PstI restriction site. I am assuming that you can't go onto digesting with the PstI now because during ligation this may religate. So, then I do my ligation of my insert into my vector. Then, I can do a one hour digest with PstI. If I heat inactivate the PstI (80 degrees for 20 minutes), can i do the transformation directly with this product or will i need to clean up the mixture first? Would this be done by running on a gel again, extracting band and then purifying? I am terrified of precipitation it never seems to work for me, but so far my ligations have been successful without doing it!
This method sounds great, I love this site sooo much!!!