Originally Posted by clevermizo
OK Here's the deal:
According to Invitrogen tech support, 1 uL of TOPO-TA vector contains 10 ng of DNA. Furthermore, the best molar ratio of PCR Product: TOPO vector is actually 1:1. I always do a calculation first and see how many ng of PCR product I need to use in the TOPO reaction, and I get almost entirely white colonies after blue/white selection.
Here's what I do:
Do PCR. If PCR is clean, just do PCR cleanup (Qiagen) to get rid of salts/dNTPs/primers, etc. If it's dirty I do gel extraction. I measure DNA concentration by A260 or run it out on a gel if it's too low.
Then before I do anything else I calculate: (10)*(kb PCR)/(3.9 kb TOPO) = __ng PCR product for reaction. Once I know that I scale up appropriately to a 10-25 reaction volume for adding 3'-A overhangs with Taq (I always use high fidelity polymerases for cloning). A simple 10 min @ 72C always does the trick.
If I need x ng PCR product for TOPO reaction, I plan my 3'A reaction to contain x ng/uL PCR product. This way, I add exactly 1 uL and it simplifies my life. I mean, you do whatever you want, but I highly recommend calculating how much PCR product you need to add to the TOPO reaction before doing it, it really helps out. I always transform into regular, cheap DH5alpha. No need for anything fancy (although Invitrogen does force the TOP10 on you).
Using too much PCR product in the TOPO kit is a bad idea. I've had to dilute PCR product 100-fold. It's not going to disappear. Less is definitely better than more. Too much PCR product will pretty much ensure you have all incorrect clones.
I realize this thread is long dead but I want to give this a try nevertheless!
I have just cloned for the first time and obtained plenty of clones, but after PCR screening it appears that my bands give a fragment shorter than the gene I wanted to clone!
I added 4 microlitre of PCR product to 1 microlitre of vector. The reason I did this is because I cut a specific band in a gel and purified it, leaving me with (so I thought) not very much DNA, so I opted to add the maximum amount possible for transformation.
In one of your posts you said that "Too much PCR product will pretty much ensure you have all incorrect clones". Could you please elaborate on this? Is it possible that only part of my gene was inserted into the vector? I am still a bit puzzled why all my obtained fragments are only 500 bases long when I added a purified 1000 base gene to the vector. I am hoping that the incorrect Ratio of vector/ PCR product could be the problem?
Thank you very much in advance