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How to set up simultaneous digestion?

plasmid vector digestion

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#1 Guf

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Posted 01 February 2013 - 01:36 AM

Hello, I need your recommendation. I need to digest my plasmid and PCR product with 2 different endonucleases, BlpI (10 000 U/ml) and HindIII (100 000 U/ml). Both of them are from NEB.

According to Double Digest Finder (https://www.neb.com/...e-digest-finder) I should perform digestion in NEBuffer 2 at 37 °C.

And my question is how to set up the reaction?

I found that restriction mixture composition:

DNA - up to 1 ug
Buffer (10x) - 2 ul
Enzyme 1 (10 U/ul) - 1 ul
Enzyme 2 (10 U/ul) - 1 ul
H2O - up to 20 ul

What do you thing? Second question is: How long to digest it? Are 2 hours sufficient for digestion?

Is it possible to dilute HindIII (100 000 U/ml) to working concentration (10 000 U/ml) in water? Or I should to choose something else, for example NEBuffer?

BlpI can not be heat inactivated so after digestion I have to purify the mixture. Is it better to purify it with PCR purification kit or use Gel extraction kit? Thank you :-)

#2 phage434

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Posted 01 February 2013 - 06:20 AM

Don't dilute your enzyme at all, just use 1 ul as it is provided. You should limit the volume of your DNA to less than 5 ul in this reaction, to avoid issues with contaminated DNA inhibiting the reaction. Either column or gel purification will work. Make sure you have purified your PCR product before digestion - otherwise your PCR enzymes and dNTPs will extend the cut DNA fragment and blunt the ends. Next time, choose enzymes that can be heat killed.

#3 Guf

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Posted 04 February 2013 - 07:14 AM

I tried the digestion for 2 hours with this mastermix:

DNA: 1000 ng (10,9 ul)
NEBuffer 2: 2 ul
BlpI (10 000 U/ml): 1 ul
HindIII (100 000 U/ml): 1ul
H2O: 5,1 ul

and this is my result:

http://imageshack.us.../cleavagen.png/

According to NEBcutter there should be 2 fragments: 7000 bp and 90 bp... but I cant see the the 90 bp fragment :-(

What is wrong? Thank you.

#4 phage434

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Posted 04 February 2013 - 07:48 AM

The intensity of a 90 bp band will be about 80x lower than the intensity of a 7000 bp fragment cut from the same plasmid. You probably are not seeing it just because it is so dim.





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