Which enzyme is to be digested first in sequential RE? - In vector preparation for cloning... (Jan/09/2007 )
Dear All,
For preparing the vector for the cloning I need to digest my plasmid with AfeI (Eco47III) and XbaI.
As they have different buffers, so which enzyme is to be digested first. Is it necessary to gel purify the plamid after first cut? How long do u incubate the RE digestion for preparing the vector? I am going to use this cut plasmid (AfeI and XbaI) many times in future, so how much reaction volume I can take?
Can anybody tell me the protocol and are there anything to be taken care of while doing sequential RE? (tricks and tips are welcome!!!)
Thanks in advance!!!
Hi, Niraj.
You can start with either one of them. After the first digest, you need to perform ethanol precipitation before you begin your second digestion with another buffer. Later, after your second digest, if possible, heat inactivate your enzyme and run it on gel and do gel extraction.
Regarding the time for digestion, it depends with the amount of DNA you are using in the digest. Normally, you can try to incubate it for 2 hours and run a small amount on gel together with your uncut DNA. If your DNA is not completely cut after the 2 hours digest, then you can incubate them overnight.
There are no specific tricks and tips for digestion. Just follow the protocol comes with the enzymes.
Good luck. 
Hi Virus Fan,
Does it mean that I need to run the digest on gel after the first direst? Or directly ehanol precipitate it? Which salted ethanol will work? For how long and what temp I need to keep it?
Thanks...
I don't know the provider of your enzymes, usually there are several buffers where enzymes are more or less efficient.
I would start to digest XbaI in NEBuffer 4 (75 % efficiency) and then check it's digested (just a small aliquot) and then add Afe I (100% efficiency).
or check the compatibility of the buffers from your provider.
no purfication steps needed.
i would ethanol precipitate after the first digest and then continue with the second digest. Run the digested (double digested) product on gel along with uncut to compare if the digestion worked efficiently.
Do I need to directly add the salted ethanol after first digest and keep it at -70C? How long do I need to keep it at -70C?
sorry for a stupid query.. but why wud someone do a sequential digest..? can't u do a simultaneous 2 enzyme digestion? double digest? whats the purpose for sequential may i know?
thkssss!
sorry for a stupid query.. but why wud someone do a sequential digest..? can't u do a simultaneous 2 enzyme digestion? double digest? whats the purpose for sequential may i know?
thkssss!
the problem is that the two enzymes are not working in the same buffer.
so usually you try to find a buffer where both enzymes work, even if one works a little less than in it's appropriate buffer.
you digest first the one which is not in the right buffer (usually it still works fine, and you don't see anymore non digested plasmid if you increase the time of digestion).
Then you check that it is digested by putting a drop on a agarose gel.
and then you pursuie digestion with the second enzyme.
If you can't find a buffer where both works, you can try to digest first in the buffer which contains the less salt, then add salt and digest with the second.
If really the two buffers are different, then you have to digest with the first RE and then purify, and then digest with the second RE in the right buffer.