restriction direct to ligation? - skip clean up? (Jul/27/2006 )
I am having a lot of troubles getting my cloning to work...and so I am just trying to get single digested vector to religate and succussfully transform
What I am going to do is check the gel purified linear vector against just cut without cleanup vector.....is this pointless....do I have to do some kind of clean up before the ligation
I always gel purify my linearized vector and my restriction fragment insert before ligating...
The competition is on!
RE vs T4 ligase- who wants to bet?
RE vs T4 ligase- who wants to bet?
I dont get what do u mean by RE vs T4 ligase.
I always purify my digested vector and fragment thru gel before ligating.
okay...I know it sounds crazy not to purify...but I am just concerned that it is something about my purification that is inhibiting my ligation...and so I wanted to try not cleaning....
what I ended up trying is just a spin column to remove the protein (after inactivation)...I will let you know how it goes
have a good one
I think that it is important to do the gel purification in order to ensure a "pure" final product. Without the gel purification, it is difficult to ensure that you have your product of interest rather than undigested origional product. The gel purification step can cause problems in ligation though. One piece of advice I can give you is to minimize the amount of time the DNA is exposed to UV while you are cutting out the bands. If you expose the DNA to UV to long, you will ruin your DNA and thus your ligation. Also, instead running your gel with EtBr, stain the gel with a quick soak and wash instead. This will also help the integrity of your DNA. Also, make sure you are not melting your gel and too high of temperature. I think 55 C is the standard. It could be your ligation conditions though and not your gel purification. It could be something as simple as a bad enzyme or expired buffer. Are you including a control ligation? You can try different conditions and lengths of ligation as well. (Example: 1 hour roomtemp, 16 C overnight). Hope this helps
"I know it sounds crazy not to purify...but I am just concerned that it is something about my purification that is inhibiting my ligation..."
boy have I been down that road...I tried purification via multiple methods when it became apparent that something with my columns seemed to be tied to inhibition of the ligations...not to sound like a total nutjob, but that solved the problem
spun the band through glass wool, through a 1ml pipet-tip filter, using different elution buffers; apparently there is a method involving snap-freezing the band but I haven't tried it? good luck
Thank you everyone for your advice...I am seriously about to LOOOSE IT!!
I have control ligations...and looking back...I think that I have probably exposed it to UV too long....does it just result in degradation?? Also I tried a column out of desperation and I am not too sure about it.
I am going to come in on the weekend and try another labs reagents....the only thing that will be mine is the plasmid...hopefully the TE buffer which it is involved in won't hurt things too much
please send me all your positive vibes
UV will cause not only lesions in the DNA, but it can also distort the structure which can also cause serious problems. Good luck!
Homebrew has sent me an article about mixing guanosine in the TBE buffer that seems to protect DNA a lot during exposure to UV. bad thing i can't find guanosine in this lab...