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pcr product digestion problem

digestion problem

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#1 ntknr

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Posted 15 November 2012 - 02:29 AM

hello

I have an insert is 200 bp.it is a pcr product. I did pcr purification and it is 138 ng/ul and 158 ng/ul. my vector is 6000 bp and 580 ng/ ul. I want to clonning but I am not . My restricition digestion enzymes NotI and PacI (FERMANTAS 10u/ul). I can not set of the amount of enzyme and vector and ınsert. how do I adjust the amount of reaction? fermantas is recommend

10X Buffer G
4-fold excess of NotI
4-fold excess of PacI
Incubate at 37 but ı can't understand.please help me.

#2 bob1

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Posted 15 November 2012 - 12:04 PM

How much DNA do you want to digest?

Use n (amount you want) =concentration x volume.

Check that Not1 and Pac1 are compatible for double digest and using appropriate buffer.

#3 John Forsberg

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Posted 15 November 2012 - 06:00 PM

Those are the conditions recommended by the ThermoScientific DoubleDigest calculator. It recommends adding 4x the normal amount of restriction enzyme, based on the idea that you're decreasing the activity of the enzymes for the sake of doing both cuts simultaneously. Since NotI and PacI will not both cut at 100% in any buffer (PacI has it's own unique buffer, and NotI only cuts at 100% in their Buffer "O"), it's suggesting that you add 4x as much NotI and PacI as you normally would. Alternatively, they recommend using the unique buffer for PacI, but still using 2x as much NotI and 4x as much PacI (which makes no sense to me because you'd expect PacI to be 100% active in its own buffer).

From their PCR product digestion protocol PDFs, they recommend 10-20 units of NotI to digest 0.1-0.5 ug of PCR product. So you would need to add 4x that much (40-80 units) for 0.1-0.5 ug of PCR product in Buffer G. They recommend the same amounts for PacI, so you'd need to add 40-80 units of both enzymes for 0.1-0.5 ug of DNA.

For your vector, from the main enzyme datasheets, it looks like they recommend 10-20 units per 0.5-1 ug of DNA. So you would need to use 40-80 units per 0.5-1 ug of vector DNA for that reaction in Buffer G.

Because of the poor activity of both of the enzymes in that buffer, I wouldn't try to do a double-digest with the enzymes you have. If you can't change the restriction sites you're using, an alternative is using their FastDigest enzymes, which are all supposed to work in the same buffer (according to their site). It looks like they have FastDigest versions of both NotI and PacI.

Otherwise, I'd try to change the enzymes to ones that can perform a double-digest in the same buffer. Trying to cut in a buffer that neither enzyme likes is asking for trouble, in my opinion.

#4 ntknr

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Posted 07 December 2012 - 04:45 AM

thanks for your information. ı did 20 unit enzyme for vector.because my teacher said to me not use for more enzymes.but ı changed the reaction conditions. 4 hour 37 centigrade degree and 12 hours 25 centigrade degree and 10 minute 65 centigrade degree. I use 2.5 ug vector for 20 unıt enzyme. Now I will do ligation but I don't know How ı adjust amount of insert vector. I tried to use ligation calculater system but ıt was said ... ng insert use. but I don't know after gel exraction How much vector and insert I have. I send the gel extraction picture.according to picture Could you help me ligation set up vector ınsert amount?

#5 John Forsberg

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Posted 07 December 2012 - 04:40 PM

We usually use a NanoDrop to quantify our DNA after cleanups/gel extractions. I would normally hope to get ~50-100 ng/µl for a vector gel extraction, and maybe 20-50 ng/µl for my inserts (if 5 µl of the PCR product looks clean with a single band on the gel, I'll just do a PCR cleanup). How did you quantify the DNA from your samples earlier? You said you had "pcr purification and it is 138 ng/ul and 158 ng/ul. my vector is 6000 bp and 580 ng/ ul"?

For a ligation, I would mix roughly 100 ng total DNA of vector+insert in 10 µl total volume for the reaction. For a first try, most people seem to suggest a ratio of 3:1 molar excess of insert over vector. If that doesn't work, but you're sure your enzymes are cutting, you can try changing the ratio lower or higher (from 1:1 to maybe 10:1 insert:vector).

For that calculation: ng insert = 3*(ng vector)(bp insert)/(bp vector)

In your cloning, with a 200 bp insert, 6000 bp vector, I would use: ng insert = 3*(90 ng vector)(200 bp)/(6000 bp) = 9 ng insert with 90 ng vector. (I roughly estimate the amount of vector to use by taking the length of the vector (6000) and add 3x the length of the insert (600) (total 6600), then figure out what % of the total the vector is (6000/6600 is ~90%)) There may be an easier way to calculate it, but this is how I do it.

I have also been told that if you run your DNAs on a gel, if you can see the band at a given volume loaded, then you can use that amount in a ligation (whether it is the insert or the vector). So if you loaded 2 µl of your insert and vector DNAs, and can faintly see bands for both of them, then that's a good amount of DNA of each to try in a ligation. I've never done my ligations that way, but if you have no way to determine the concentration of DNA in your samples, then that might be worth trying.

Edited by John Forsberg, 07 December 2012 - 04:42 PM.





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