Overtreatment by dephosphorylating enzymes - (Jul/24/2007 )
Hi!
This is somewhat of a crisis for me: I am not sure if there is actually something like "overtreating" your vector with a dephosphorylising enzyme.
I need to prepare a vector for library construction, but the back-ground after normal digestion & SAP-treatment is simply too high. Background was reduced immensely since I started doing my SAP-treatments in two consecutive steps (3ul in a 50ul reaction, 37degrees, 60min, followed by deactivation ...followed by 2ul in the same reaction mixture, same procedure). However, my libraries do not have nearly enough colony forming units after transformation in 10^10 electrocompetent cells.
I do not believe that the problem lies with the transformation procedure however.
Is there anybody that knows if it is possible to actually do harm to your vector by submitting it to such intence dephosphorylation procedures?
Any insight into the matter would be highly appreciated!
absolutely overphosphorylation can be a problem
if your background is that high, there are two things you might try: use a different de-phosphorylating enzyme (I like anarctic phosphatase from NEB; everyone has their favorite), or try to increase your vector digestion efficiency. if there is even a little bit of undigested or nicked vector in the tube, it will usually transform with much, much higher efficiency than your ligation product and this could be the source of your high background
good luck
This is a common problem with dephosphorylating enzymes. You should try to design your reactions to avoid their use. Use vectors which are cut twice with different enzymes. Or make your vector backbone with PCR as we do -- this eliminates the uncut circular and single cut vector background. We now do the PCR with biotin labeled primers, and remove the undigested PCR products and short ends with a streptavidin purification step, leaving only correctly cut linear vector backbone.
yes, it nibbles away at the ends.
You could try doing a time course to see what time will leave your vector dephos without actually harming it. we did this, and found that 5 minutes was all we needed to dephos our vector. though, in different hands, this time will vary.
V
if your background is that high, there are two things you might try: use a different de-phosphorylating enzyme (I like anarctic phosphatase from NEB; everyone has their favorite), or try to increase your vector digestion efficiency. if there is even a little bit of undigested or nicked vector in the tube, it will usually transform with much, much higher efficiency than your ligation product and this could be the source of your high background
good luck
Yes, the enzyme that I am using (SfiI) is rather exceptional in that it exists in solution as a tetramer and it appears to interact with two copies of it's recognition sequence before it can cleave DNA. It's primary reaction is then to cut both strands at both sites in a concerted process. (from Lois M. Wentzell, Timothy J. Nobbs and Stephen E. Halford, Mol. Biol. 248:581-595 (1995)).
Unfortunately the enzyme is not cis-specific for these binding sites, but cleaves trans-sites equally. You can imagine that by the end of a digestion, the enzyme will struggle to find a second recognition sequence to bind to - this makes it very difficult to establish complete digestion.
However, I am trying to differentiate between fully & partially digsted products by gelelectrophoreses & excision. Will try out another phosphatase as well.
Thank you!
Hi Phage434!
One of the beauties of the enzyme that I'm using is that it can create non-complementary sticky ends due to its unique recognition sequence - therefore two-enzyme digestions are not necessary.
Your suggestion about the vector backbone however seems like a very good alternative to me. Could you inform me of what enzyme you use to construct yours, and what the largest vectors were that you've created in this way? Also, I am not sure: How do you differentiate between full length PCR products and early termination products?
Would it be possible to direct me to a protocol for creating vectors in the way that you do?
Much appreciated!
You could try doing a time course to see what time will leave your vector dephos without actually harming it. we did this, and found that 5 minutes was all we needed to dephos our vector. though, in different hands, this time will vary.
V
Thanx vetticus3!
A clear-cut answer to confirm my suspicion - a good thing, because at least now I can start trouble shooting at something concrete! Will try out the 5min step first of all.
Regards
TabulaRasa
I'm still developing the PCR backbone approach, but check here:
http://openwetware.org/wiki/Streptavidin_p...f_DNA_fragments
Note especially the diagnostic procedures for testing the quality of the results.