lentiCRISPR v2 plasmid smear on gel after digest

[Sarah S.]

Hello,

We are currently having problems with cloning our gRNAs into the pLentiCRISPR v2 plasmid, specifically, we see smearing on the gel when we run digested plasmid out.  Below is the protocol we have been using: 

After receiving the bacterial stab from Addgene we streaked it out onto an Amp plate and grew at 30 degrees overnight.  We then picked three colonies, grew those up, preformed maxipreps, and then digested with BsmBI.  Upon digestion with BsmBI we see two bands, one around 2kb and the other around 13kb, however, most of the lane is a smear. 

We have tried this now with a) multiple clones, b) two different stabs from Addgene, and c) several different tubes of enzyme and/or digestion buffer.  We use the NEB BsmBI in Buffer 3.1 at 55 degrees.  

When cut alongside pLentiCRISPR v1, we see two nice bands of V1 but a smear with V2, suggesting that this is a plasmid specific problem.  

Has anyone else had this problem?  If so, how did you resolve it?  Any suggestions for fixing this would be much appreciated!

Thanks!

Sarah  

[Neville Sanjana]

Hi Sarah,

Sorry to hear about your troubles. It is puzzling, especially given your success with lentiCRISPRv1. (The digestion/removal of the filler/dummy insert is identical between the two vectors.)

Given that you see the 2kb filler pop out, you might try gel extracting the larger band and proceeding with cloning. My guess is that this will work. If you are concerned, you can always sequence key elements after cloning in your guide sequences.

Best wishes,

Neville

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[Duško Lainšček]

dear,

i am having simillar problems.After digestion with BsmBI I get only smear.I allready tried everything; less enzyme, shorter time etc...any suggestions?

Thank you

Best

Dusko

[Phil Abbosh]

Hello
I had the same smear with the NEB BsmBI and when i gel purified the big band it did not work after several tries and modifications. i digested for a shorter time (10 min) and had less smearing. however, shorter digest times dont completely cut the plasmid, so unless you resolve for a long time on agarose, you will have background colonies.  to overcome, you can resolve uncut pLCV2 alongside in an adjacent lane, and only purify out the band when the digested and undigested bands have migrated to clearly different distances.  then, any uncut plasmid in your digested lane will be left in the gel and you will purify only the cut plasmid.

 when ran out of NEB enzyme, i switched to the thermo-scientific enzyme, it worked great without all the trouble.  you have to add DTT separately but it's easy and now i get lots of colonies.

phil

[Abhay Uthale]

Hello,

I am also facing the same problem with lentiCRISPRv2, where I am seeing smearing of my plasmid and also I am not getting 13kb fragment but could see 2kb filler fragment.

The protocol I followed was same as the Sarah only thing that is different is I am using Esp3I (isoschizomer of BsmbI)  enzyme from thermo fischer with 37 deggee incubation for 1 Hr.

I ran several different variations where I checked for nuclease contamination but it seems there is no nuclease contamination in the buffer, enzyme & water. I also ran digestion reaction with the plasmid which do not have this Esp3I site. I am uploading gel picture where,

Lane 1: 10kb ladder

Lane 2: Undigested pcDNA3.1

Lane 3: Digested pcDNA3.1

Lane 4: Undigested lentiCRISPR v2

Lane 5: Digested lentiCRISPR v2

[Carter]

Thermo Fast Digest BsmBI works well. I've never had smearing and it is fully digested. 

[Yan Cui]

Stbl3 strain, which is used by Addgene for Lentiviral vectors, contains high level endonuclease activity.

If you do the mini-prep using Qiagen QIAprep Spin Miniprep Kit, you should include an additional washing step using PB buffer before washing using PE buffer.

This tip also applies to other similar method for plasmid prep.

You should include a digestion ctr that incubating the plasmid at 37C (no RE inside, just put the plasmid into your RE buffer). If the smear shows up after incubation at 37C, then you need make new prep.

If not, then it could be due to the bad quality of BsmBI. I used BsmBI from fermentas, did not get problem. 

[Alex Ling]

I concur with this reply. Stbl cells are endA+, and some of that DNase makes it through into the eluate from miniprep columns-- I found this out first hand the bad way. A quick fix would be to inactivate the DNase by phenol-chloroform or gel extraction.

[porquet...@gmail.com]

Hi everyone,

I had the same problem with this plasmid (pLenticrisprv2). I fixed it with the use of C3040 competent cells (NEB). In addition, I use ALWAYS 30°C as temperature for cloning, miniprep or maxiprep.

I hope this reponse will be useful.