double digestion with BamHI and HindIII - (Jan/04/2007 )

Hi all,

I am trying to double digest my vector in one step using enzymes BamHI and HindIII from New England Biolabs. I want to know if I can do double digestion with these enzymes using a single buffer. The activity of HindIII is 100% with buffer 2, and activity of BamHI is also 100% in presence of buffer 2, but the company recommends using a buffer specific for BamHI.

I tried to do sequential digestion using two different buffers but I was not successful.

Thanx for your help.

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BamHI and Hind3 in NEB buffer 2
yeah that is fine.

Though I am concern about the sequential digest not working. Did you give the digest enough time? How much DNA and how much enzyme was used?

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I use buffer 2 for hind2 and bamh1 double digests and it usually works, although NEB recommends the sequential digest.

As perneseblue wrote, I too am curious as to y ur sequential digest didnt work.

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yes it's work with me but need more time and enzyme you can increase them

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I double digest Bam and HindIII together in one buffer B from Roche. I dont know why but old buffers from NEB dont work here .I f i have two enzymes first i try to establish good buffer that works for them both and if that doesnt work i go step by step digestion. Try also buffers from another company if you have. In my experience that works even better sometimes. Leave them overnight at 37.

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Thanks for all your advice,

This is how I did my sequential digests:
I first digested my vector with HIndIII for 4 hrs at 37C. I used 1ul enzyme in 5ul DNA. Following this digestion I purified my sample and did subsequent digestion with BamHI for 4hrs at 37C using 2ul enzyme and 30ul purifies HindIII digested DNA. After this I again purified my sample and then ran on agarose gel.

I am not sure is it feasible to do purification twice??????

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Thanks... but the details are not quite complete. I will try to be more percise
How much DNA
How much Enzyme
How much Buffer
How much BSA
Total volume of digest.

eg
5ul DNA (~5ug DNA)
1ul Hind 3
2ul Buffer 2
0.5ul BSA
11.5ul water
Total volume 20ul

If the total digest volume is too small, the glycerol (from the enzyme concentrate) content will became inhibitory to the restriction enzyme activity.

Feasibilty of doing two column purification would depend on the quantity of DNA you have, DNA szie (columns have cut off points) and the time you are willing to spend. However in this case, there is no need to removed the first restriction enzyme. HInd3 won't do anything bad. It is a rather well behaved enzyme.

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Here are the details:
5ul DNA
1ul Hind 3
2ul Buffer 2
2ul BSA
10ul water
Total volume 20ul - incubation at 37C for 4 hrs with Hind3


For BamHI digestion:
30ul DNA
2ul BamHI
5ul BamHI Buffer
5ul BSA
8ul water
Total volume 50ul - incubation 37C for 4 hrs

I am not sure of the DNA conc in ug......and I am using pET 30 vector which is ligated with my insert (sorry I forgot to mention this earlier)

Thanks again.....

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with BamHI you use 30ul DNA. maybe its too much? if you dont know how much DNA is there how can you know if the enzyme is enough. try to decrease DNA or increase enzyme. but be careful with glycerol as mentioned before. you can increase the overall volume of the reaction to avoid this problem.

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BSA is a 100x stosk sol. , so u r using it as if it was 10x. So u need to adjust it.

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