ligation of pcr product into expression vector
Posted 04 October 2011 - 11:53 PM
Today I used my PCR product (that was cleaned up) to ligate into PET expression...just want to see if I went about it the right way:
Cut my vector with xho1/ecor1 and corresponding buffer and water up to a volume of 20uL...kept at 37 degrees for around an hour
cut my pcr product with same enzymes/buffer/water up to a volume of 20uL and kept at 37 degrees for an hour
ran a gel to see if dna was present..it was so then took
7uL of insert
1uL of vector
and ran one without insert (substituted with water)...
was that right? in parallel I also did a gel purification exp and ligated with simliar amounts...but just want to know if the pcr product procedure I did was correct?
Posted 05 October 2011 - 06:04 AM
Unless you heat inactivated your enzymes, you will probably have a problem ligating vector & insert without gel purification. Xho and EcoRI can both be heat inactivated at 65 degrees for 20 min. Without that, the enzymes will just re-cut any ligated product.
As far as you ligation goes, I won't harp on this because everybody has their own Voodoo about ligations, but I was taught to treat a ligation like any chemical reaction (which it is). If you were setting up a reaction in a chemistry class, would you just throw in a few mLs of this and a dash of that? Probably not. In chemistry, you calculate the concentrations of the reactants and you quantitate everything. So, I would recommend calculating the concentration of your cut vector and insert (preferrably by EtBr gel/compare to ladder bands) and then set up a ligation with a 3:1 ratio of insert to vector, using a total of 50-100 ng of DNA per 20 uL ligation. Like I said, my protocol is not everyone's protocol, but it works well for me.
Last question about your proceedure, but are you able to tell by gel if your PCR product has been cut? if would imagine that you simply added XhoI and EcoRI tails to your PCR primers, which would make it impossible to tell if they have cut properly. If you didn't give yourself some extra sequence outside of the restriction enzyme sites in your primers, your cutting efficiency can go way down with no way to know until your ligation doesn't work. If you are unsure, you can post your primer sequences for us to check or refer to the NEB website with regard to enzyme efficiencies here: http://www.neb.com/n...nucleotides.asp
Best of Luck.
Posted 05 October 2011 - 02:01 PM
You should be calculating the ratio based on length of insert relative to the vector, as obviously 10 ng of a short insert will have more copies of that piece of DNA than a large vector will for the same mass. You can calculate the required ration of insert : vector in ng using the following formula:
amount of insert (ng) = ratio x (insert length (bp) / vector length (bp)) x amount of vector (ng).
For example if you want to use 20 ng of a vector which is 2000 bp, and ligate in a 200 bp insert at a 3:1 ratio, you would use
insert= 3 x (200/2000) x 20
= 3 x 0.1 x 20
= 6 ng
Posted 05 October 2011 - 02:28 PM
For the gel purification technique I usually I use the calc. regarding how much insert to vecotr I must use after seeing them on a gel..but then i was asked "did you use as much insert as possible" and was told once b4 it was bettter to use as much insert as pssible hence I used 7uL (relative to my vector band, my insert band was rather faint)...
but i still don't get how to ligate a pcr product into a expression vector sorry..i have cleaned up my pcr product but in terms of how much of each thing to use and how much volume to make up totally im still confused.......am i suppse to gel purify it after my digest is over...i hv looked and looked for a protocol on directly ligating a pcr product into an expression vector but have no luck soo if someone could pls help wiht a good site or in general give me a run down on wat i need to set up it would be much much MUCH appreciated