Do you discard phosphatase before ligation? - (Jan/31/2008 )
Hi all,
I was wondering if I should do any step between dephosphorylation of my vector (single RE cut) and ligation with insert; either to remove CIP or to eliminate the 4uL EDTA 50mM I use to stop it (is T4 DNA ligase sensitive to EDTA?). This ligation has never been too successful around here...
Thanks!
The reason CIP and BAP are used at elevated temperatures is that the exonuclease contaminants are less active at high temps. You should never use excess enzyme. You must clean the DNA before attempting to ligate. SAP-type dephosphorylases do not have this problem and can be used at 37C during the restrictiction cut.
Clean DNA always ligates better.
I was wondering if I should do any step between dephosphorylation of my vector (single RE cut) and ligation with insert; either to remove CIP or to eliminate the 4uL EDTA 50mM I use to stop it (is T4 DNA ligase sensitive to EDTA?). This ligation has never been too successful around here...
Thanks!
my recommendation: check out NEBs website- theres loads of useful information there. among it the answer to your question:
Q1: What are some potential problems with the ligation reaction using T4 DNA Ligase that can lead to transformation failure?
A1: * Ligation failed because there was no ATP or Mg2+. Use the supplied buffer or add ATP to a compatible buffer. The ATP in buffers older than one year may have degraded enough to cause problems. When supplementing with ATP, be sure to use ribo ATP as deoxyribo ATP will not work.
* Ligation failed due to high salt or EDTA in the reaction. Clean up the DNA.
http://www.neb.com/nebecomm/products/faqproductM0202.asp#339
by the way... why do you use EDTA at all??? just gelpurify after treatment.
little reminder: T4 ligase works in all four NEBuffers and PNK buffer. so no need for cleanup if u dont need to CIP/SAP...
cheers
Thanks for your input, I'm going to clean up the DNA before ligating. I'll tell you how it goes.
What about after ligation, before transformation? I've seen some protocols including a precipitation step, others don't. I'm going to try to precipitate with sodium acetate and ethanol before heat-shock, but I have so little DNA I'm afraid there won't even be a pellet.
use something like antarctic alkaline phosphatase instead of CIP, it can be heat inactivated and the DNA used directly for ligation, especially if you donot have lot of DNA.
I would, but there's none around here and we're poor 
besides, I'm not even a PhD student so I don't have the status to order something so transcendent as an enzyme 
I'll have to make do with CIP.
Anyway, there *was* a pellet! Yay!
1. Despite my preference for SAP-type anzymes, CIP and BAP work well when used properly. So if CIP is all you have, don't worry.
2. In more than 25 years of optimizing cloning reactions, I have never bothered to precipitate the ligation reaction before transformation. I ligate with 5 ng of blunt-end vector in a 5 uL reaction, transform 1 uL in 25 uL of cells, heat pulse, add 680 uL of SOC, plate 30 uL. Blunt end ligations give me an average of 300 colonies/plate, 85-90% with inserts. Numbers are higher if I use my own electro-competent cells, but commercially available are adequate.