Simple Question - (Jan/02/2009 )

I have some simple Question. If two restrcition enzymes can be used for double digestion then after First digestion does its necessary to clean and concentrate it and then do second digestion or one can skip clean and concentration step and can go for second digestion.
Similarly after digestion going to Dephosphorylation step, clean and concentration step is necessary or not.
regards

Hey

I use enzymes from NEB. For CIP treatment, just digest and in the same mix, add CIAP without any buffer. It works.

In case of a double digestion, look for compatible buffers and digest in that. In the manual, somewhere they mention the % activity of an enzyme with all buffers, choose the compatible one, where activity for both enzymes is 100%.

If u don't find a compatible buffer, u can do 3 things:

1. First digest with the buffer which has a lower conc. After first digestion is done, inc. the conc to the one req. for the second enzyme and add the second enzyme.

2. Clean the reaction up with PCR purification kit after first digestion.

3. Precipitate the DNA after first digestion with ethanol and sodium acetate.

Hope it helps.

TC

I am cutting my vector with Nde1 and BamH1 enzyme and i am getting more then two bands. It seems it has star activity.
If I first cut with Nde1 and then with BamH1 vector then i get normal two band, my insert and the the linear vector, does its becasue of star activity.......
Can you tell me
whats the criteria to use how much amount (whats the criteria for amount)of buffer and BSA is used in the reaction mixture

Hi

I get a feeling its undigested DNA in its three forms and not star activity.

Ckeck for a compatible buffer. The old NEB Bam HI isn't compatible (it used to come with its unique buffer), however the new one is compatible in buffer 3 (kindly check), the buffer supplied is NEB2, as far as I remember.

Both buffer and BSA is 1X. i generally use 0.2 to 0.3 ul of enzyme in a 20ul digest.

If it still doesn't work, just digest 3 vials with the first enzyme (i would choose bam HI), precipitate and pool and setup the second digestion.

TC

I agree with T C -- it might not be star activity. Do you know (by experiment) that the vector cut with each enzyme on its own produces a single band?

So how to get a complete digestion, either to increase the time of digestion or increase the amount of enzyme.
I usually restrict for one hr......

Hi

I prefer increasing the time. If its a check digestion (to check clones), i digest with 0.3 ul of enzyme (kindly chk standard units from the NEB vial, I am not in lab) for minimum of two hours and if its a prep digestion (for gel elution and cloning), i digest for 8-12 hours with teh same amount of enzyme in a 20ul reaction mix.

In my 5 yrs of research experience, i have never seen star activity with any of the enzymes and have used nearly 30-35 enzymes.

TC

Hello there,

I am cutting my vector with the same enzymes as you. What I do is to cut with Nde1 first (NE Buffer 4 was used) and incubate at 37deg for 2hrs before doing a PCR clean up to remove the unnecessary stuffs. I will then do an enzyme digestion using BamH1 (NE Buffer 3 used, as well as BSA) and incubate at the same conditions.