CIP with restriction digestion - (Dec/05/2005 )
I need to use CIP to lower my background on my transformation plates. I was digesting my vector with EcoRV and BamHI in BamHI buffer for 4 or 5 hours and gel-purifying. I also did single digest controls to make sure each enzyme is cutting properly. But I still get lots of vector only and vector plus ligase colonies. I am thinking of using CIP but can I add CIP directly into my restriction reaction after 4 hours and leave it there for 1 hr then do the gel-purification?
CIP will not help if the vector is uncut. If you prepare your vector by PCR rather than by miniprep, there will be very little background plasmid. Use two primers which replicate the entire plasmid, directed outward around the cloning site. Use cut sites already present on the plasmid, or add new ones to the 5' end of the primers. PCR the plasmid, cleanup, mix with your insert, digest, ligate, and transform. No background.
CIP is active on all neb buffers.
But it works better when you have a 0.5µg/µl plasmid concentration. Hence you need to digest in a first volume, during 4 to overnight time, and then dilute your prep
Cip is effective in 1. Then inactivate by 30' 65° and gel purify.
BUT... :
Activity of EcoRV in neb buffers is 100% in NEB3, 75% in neb 2 and 50% in other ones.
activity of BamHI in NEB3 is only 50%....
moreover, EcoRV activity in BamHI buffer is 0%
so i think you need to do sequential digest. First bamhi and then ecoRV (as i don't know activity of CIP in BamHI buffer)
But it works better when you have a 0.5µg/µl plasmid concentration. Hence you need to digest in a first volume, during 4 to overnight time, and then dilute your prep
Cip is effective in 1. Then inactivate by 30' 65° and gel purify.
BUT... :
Activity of EcoRV in neb buffers is 100% in NEB3, 75% in neb 2 and 50% in other ones.
activity of BamHI in NEB3 is only 50%....
moreover, EcoRV activity in BamHI buffer is 0%
so i think you need to do sequential digest. First bamhi and then ecoRV (as i don't know activity of CIP in BamHI buffer)
Fred 33 is right follow the instructions but a complement is that EcoRV is if I remember correctly a blunt end site and for thoses sites you need to go with two incubation steps
1/ 37°C for 30mn
2/56°C for 15mn
add another shot of CIP and repeat the incubation
1/ 37°C for 30mn
2/56°C for 15mn
Pesji
Last time I checked, EcoRV in BamHI buffer is 100% activity. That's what the double digest chart recommends using as well. I've always done this pair in the BamHI buffer and the EcoRV single digest control always looks fine in BamHI buffer. I wasn't sure if you mean to dephosphorylate a blunt end site I need to do 2 step incubation when using CIP? I couldn't find that information in the NEB catalog.
Well yes of course you need to dephosphorylate blunt end also it's the same but tougher like the 5' recessed termini which also need this two step incubation at least with the Promega CIP enzyme
You can find this on the NEB FAQ's
Q8: Why do some protocols recommend using CIP at 50°C and some recommend 37°C?
A8: It has been shown that DNA fragments with 3´ extensions or blunt ends are slightly more difficult to dephosphorylate with CIP than those fragments with 5´ extensions. CIP works slightly better on 3´ extensions and blunt-ends at 50°C. We have found that the difference in efficiency is about 20-25%. Some protocols also recommend the addition of more CIP for these difficult ends. For the sake of simplicity, we recommend 37°C and an enzyme concentration of 0.5 unit/µg in a 10 µl reaction for all types of ends.
The adress oft NEB FAQhe FAQ is
Pesji
