I am using Sac I (NEB BIO labs) RO 156S 2000 units - (20,000 U/ml)). How many microliters will need for 20 Microliter Restriction Reaction? (2 microgram of DNA). Sac Data sheet didnt give proper instruction? and also they were provided 10x buffer and 100x BSA .
my Reaction setup:
DNA - 6 microliters(2 microgram)
Mul1 - 1 microliter
Sac I - 1 microliter
NEB Buffer- 2 microliters
BSA - 0.5 microliter
NF.Water- 9.5 microliters
Total Reaction volume - 20 microliter
NEB Buffer concentration is now 1x . Is it correct can i change that one
BSA concentration is 2.5x . what is the use of BSA
Normally other researchers are used 1x concentration (NEB Buffer and BSA ) or other concentration
As you mention 20,000U/ml, this corresponds to 20U/µl. I guess 10U is sufficient to cut 1-2µg DNA. So I would use 0.5µl(10U) first and if you want use 1µl(20U) in a second individual reaction. Is BSA required? then dilute the required amount to the final concentration(If 1X BSA needed then take 0.2µl from 10X for your 20µl reaction).
I agree with Ananda, 10U of enzyme should be enough for such a reaction. Regarding the BSA, this can make the reaction conditions better. An interesting article on this was published in the Promega News in 1996: http://www.promega.de/~/media/files/resources/promega%20notes/59/bsa%20and%20restriction%20enzyme%20digestions.pdf
Senthil,
Its always advisable to final concentration to 1X so buffer is right and BSA should be 0.2µl.
Your reaction setup looks fine though you have excess BSA (which dont harm reactions but not sure in this case) but you should check the double digest compatibility and when I checked for Sac I and Mlu I(you have mentioned Mul I), Mlu I has 50% activity in buffer 4 so you need to optimise the reaction or should go for sequential digestion. Use the following link
https://www.neb.com/tools-and-resources/interactive-tools/double-digest-finder
Neb has provided this:
Unit Definition
One unit is defined as the amount of enzyme required to digest 1 µg of λ DNA (HindIII digest) in 1 hour at 37°C in a total reaction volume of 50 µl.
So are you expecting more information than this or looking for specific information?
I searched role of BSA for you, which is available on NEB.
"Bovine Serum Albumin (BSA) is supplied with some restriction enzymes to prevent adhesion of the enzyme to reaction tubes and pipette surfaces. BSA also stabilizes some proteins during incubation."
https://www.neb.com/products/b9001-bsa
FAQ: What effect does BSA have on the performance of NEB’s restriction enzymes when included in the new buffers?
NEB has not determined any negative effects when including BSA in the reaction buffer. In some instances, activity may be enhanced.
https://www.neb.com/faqs/2013/02/28/what-effect-does-bsa-have-on-the-performance-of-neb-s-restriction-enzymes-when-included-in-the-ne
I guess you got answers to all your questions. My suggestions is, set up the reaction as above with all components at 1X, do a time dependent digestion (set up a 50 uL reaction and draw 3-5 ul at 60, 90, 120 minutes and asses the digestion the agarose gel). If the gel suggests that you are not getting complete digestions then go for sequential digestion (digest with Sac I and purify the DNA to remove the salts and then setup digestion with Mul I) and confirm with gel analysis.
There could be some typos in this long suggestion, please dont mind.
Thank you very much Mr.Ananda, Mr.Christian,Mr.Yogesha.
Another one doubt, I have fermentas T4 DNA Ligase and ligase buffer. Can i use this one or compulsory i use NEB T4 DNA Ligase. Is there any buffer interrupt my Ligase reaction.
are you kidding senthil,please read the data sheet once you get any material. you should not play around with buffers like this. the QC of any company have standerdized their product with great effort. so simply follow as given. never use pfu buffer to Taq or fermentas buffer to neb! you may find difference by reading the buffer composition.
Please talk to some one in your department who has done cloning before so you could get all doubts clarified.
sorry Mr.Ananda, Mr.Yogesha.. , i Know every company have their own products but i didn't get correct result after my transformation.. thats way i have lot of doubts in every process. Above protocol is our labmate stranded protocol. Even though i have doubt about eppendorf tubes 1.5ml (using Transformation). Can i use PCR tubes for transformation (500 Microliters).
Mr.Ananda
i was used NEB restriction enzyme and buffer for my Restriction reaction, after Restriction arrest 65 c for 20 minutes , i used fermentas Ligase enzyme and ligase buffer for my ligation reaction.( currently we don't have NEB T4 DNA Ligase).
You can use pcr tube and even it's better as it has thinner wall and heat transfer better. I suggest to do column or gel clean up before setting the ligation. Fermentas ligase fine with its buffer. It's kind of similar To NEB I guess for at least for those I have checked.
Can you tell the whole story briefly. What do you mean you didn't get correct results? If you could post exact protocol you have used may be some of us could suggest improvements or some thing. Everyone in beginning goes through this type of problem.
To my knowledge it actually doesn't matter, which enzymes you use, as long as you use the provided buffers. There was even rumors about NEB buying stuff from Fermentas and re-labelling it. So don't worry about digesting with oone company and ligating with another, thats fine. For more specific advise, you need to be more specific what your problems are (or your protocol looks like).
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