About BbsI digestion

[Jing]

Hello, everyone. I'm using CRISPR/Cas Systems to manipulate my gene. I've already got the plasmid px260, px330, and px335 from Addgene. But I am stuck at the beginning. 

I use 0.3 ul BbsI (from NEB) to digest 1 ug plasmid in 20 ul system at 37C for 2 hr (as I use other enzymes from NEB). It turned out that the all of the three plasmid was cut into very small pieces (see attached file), like the enzyme works as DNase. I tried another tube of BbsI found in neighbor lab, but got same result. I have sequenced the plasmid using U6 primer, it seems correct.

I can not explain it. Is there anyone has the same problem? Is there any trick for using BbsI? Any info. is appreciated. Thanks.

Jing

[Miranda Wilson]

If you look at the NEB FAQs for BbsI you will see it's an enzyme that goes off at -20deg and is sensitive to salt. I used BpiI from Thermo instead, it recognises the same site but is more robust.

[Jing]

Thank you very much! I did store BbsI at -20C. It may be the reason.

[Lechu K.]

Hi,

I used BbsI from NEB, stored at -20 (as recommended), and it worked fine. Your DNA looks seriously chopped, never heard of an enzyme with such an (unspecific) activity (except for DNAses). I would suggest to check for potential contamination (water used for setting up rxns, etc.). Next step would be to run control reactions, skipping one component in each case, to find your what chopps your DNA.

Also try to set up a control digest with the same reagents, but with a different enzyme (eg. NdeI) to check if your plasmids are fine (NdeI should release approx. 500bp fragment if I remember correctly).

At least this it what I would do.

Good luck!

[Jing]

Thanks a lot! It's absolutely worth to do it. 

[Mohsen Basiri]

This problem usually occurs when there is DNase contamination in plasmid sample. DNase remains inactive in TE or elution buffers of miniprep. kits, but becomes active in Mg-containing buffers (e.g. digestion buffers).
You can eliminate DNase with phenol-chloroform extraction or repeat the plasmid extraction procedure with the optional washing in your miniprep kit.

[Jing]

I think you are right. I incubated the following at 37C:

1. plasmid

2. plasimd + water

3. plasmid + digestion buffer

4. plasmid + enzyme

Only #3 was chopped into pieces. I already test the buffer, it's good. So the contamination came from the plasmid. Many thanks for your help!

[Fatwa Adikusuma]

Hi All,

I used to have a problem with Bbs1, but I don't know whether my problem would be the same with yours. I found out that my problem was due to long incubation (overnight), since Bbs1 likely has star activity. I then just incubated for 30 minutes and everything was fine.

Cheers,
Fatwa

[Peter Hohenstein]

Wouldn’t heat inactivation work as well? 30 min at 65 C shouldn’t hurt a plasmid prep but I think it will kill off DNAse quite efficiently…

 

Peter

 

 

From: cri...@googlegroups.com [mailto:cri...@googlegroups.com] On Behalf Of Mohsen Basiri
Sent: 19 September 2013 18:20
To: cri...@googlegroups.com
Subject: Re: About BbsI digestion

 

This problem is usually occurs when there is DNase contamination in plasmid sample. DNase remains inactive in TE or elution buffers of miniprep. kits, but becomes active in Mg-containing buffers (i.e digestion buffers).

You can eliminate DNase with phenol-chloroform extraction or repeat the plasmid extraction procedure with the optional washing in your miniprep kit.

On Thursday, September 19, 2013 2:39:57 AM UTC+3:30, Jing wrote:

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