Degradation after Surveyor Nuclease incubation

[Yoon Seong Kim]

Dear

After transfection of sgRNA/Cas9 to knockout the target gene, we want to check indels in DSB site.

However, after Surveyor nuclease incubation, we observed no band but degradation of PCR product (low MW smearing).

This seems nothing to do with heteroduplex formation.

Has someone experienced the similar?

How you guys solve the problem?

Thanks

Yoon

[Varsha Bhargava]

I have a similar problem too Yoon.

I have not been able to sort it! 

Have you made any progress on this?

Varsha

[JP]

Your negative control shows no band, only a smear? Use less enzyme or shorten the incubation period. 

[f223...@gmail.com]

how long have you digest pcr product? 30min or more over

在 2014年9月4日星期四UTC+8上午11时49分30秒，Yoon Seong Kim写道：

[Davide Seruggia]

I had a similar experience when I did a T7EI assay on a PCR reaction containing DMSO.

I think that DMSO partially denatured DNA and so the T7 enzyme digested everything. After column purification the issue was solved.

cheers

Davide

[Yoon Seong Kim]

Dear Davide

My PCR products were column-based gel purification followed by water reconstitution.

Thanks

Yoon

[Yoon Seong Kim]

I tried several incubation time even 2 min to 15 min. But it immediately degraded PCR products.

I also used much less amount of enzyme than usually suggested, but still the same problem.

It is nothing to do with denaturation-renaturation. Just gel purified PCR product with surveyor enzyme causes degradation.

Yoon

[Yoon Seong Kim]

Both are not working.

[Andrea D'Osualdo]

I had the same problem after column-based gel purification, not sure why though...I solved the problem after PCR optimization and direct T7/surveyor without column purification. If I remember correctly there was another post suggesting the same.

Good luck,

Andrea

Sent from my iPhone

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[Phil Abbosh]

I just got surveyor to work using half the amount of recommended enzyme and half the digest time.  i use platinum taq (invitrogen) and no PCR cleanup.  have you used the + control that comes with the kit?  i found it very helpful to use the + controls to optimize reaction conditions.  still get a little smearing, but i can clearly see cut bands now. also, i see them better on PAGE than on agarose gels.

Also, note that you have to have some salt in the reaction for it to work....eluting your product in water and adding MgCl2/enhancer is probably not adequate. see pages 22 and 26 of the manufacturer instructions (attached)

[Phil Abbosh]

[Phil Abbosh]

[Phil Abbosh]

I just got surveyor to work using half the amount of recommended enzyme and half the digest time.  i use platinum taq (invitrogen) and no PCR cleanup.  have you used the + control that comes with the kit?  i found it very helpful to use the + controls to optimize reaction conditions.  still get a little smearing, but i can clearly see cut bands now. also, i see them better on PAGE than on agarose gels.

Also, note that you have to have some salt in the reaction for it to work....eluting your product in water and adding MgCl2/enhancer is probably not adequate. see pages 22 and 26 of the manufacturer instructions (attached to previous post).

[JP]

I run Surveyor on an aliquot of the PCR with no further purification in 1X complete PCR buffer (incl. Mg2+). Works great every time.

[Fatwa Adikusuma]

We no longer use T7E or Surveyor to see heteroduplex, we are convenient with just running the PCR product in polyacrylamide gel. You will be able to see the heteroduplex band that run much slower than the main band which indicates you get the mutation.

Cheers,

Fatwa 

[JP]

This is a great solution -- would you be so kind as to give more details, and possibly a picture of the gel, please?

[Fatwa Adikusuma]

There is nothing special in the protocol. After the PCR (300-600bp), re-anneal your PCR product slowly (without re-annealing works as well, but it is better with re-annealing), then run the PCR product in 12% PA gel (we use western tank) 150V for 2 hours, followed by ethidium post staining. you will also be able to see the heteroduplex band even with bigger PCR (1.5kb). In the picture you will see

lane 1 : ladder

lane 4: indel -1/+1

lane 5: indel -3/-14

lane 6: indel -21/-14

lane 7: -8 only

lane 8: -7 only

lane 9: WT control (400bp)

We have compared the T7 assay and PA gel assay and both give the same result/conclusion of heteroduplex. Save your time and money using this approach. 

Cheers,

Fatwa

[Cor]

Hi Fatwa,

That looks great, perhaps stating the obvious: this a a non-denaturing gel, right? And what running buffer did you use ?

Thanks,

Best,

Cor

[EI]

Would you get a smear above your control amplicon if there are different indels present in transfected cells?

[JP]

Thanks, Fatwa!

This is for screening clones, right? Not for bulk measurements before single cell plating?

[Yoon Seong Kim]

Hi Fatwa

Thank you for the alternative strategy.

indel -3/-14 means that you mixed two deletion mutants with WT and re-anneal then ran the gel?

-8 or -7 means you mixed either -8 or -7 mutant with WT?

little confusing.

Yoon

[Fatwa Adikusuma]

non-denaturing gel, in TBE buffer. You will get extra bands above your main band every time you run heteroduplex products. I never use this for bulk transfected cells but I guess it is possible, you will get a very messy extra bands above the main band. 

-3/-14 means that when we sequenced we found 1 allele got del of 3bp and another allele got 14bp del (no WT allele). Anyway it is not easy to get single hit only (heterogenous WT/mutant) using CRISPR.

Cheers,

Fatwa

[thomas cunningham]

I tried this directly on "bulk transfected" (non-clonal) mouse ES cells and it worked great. I see multiple bands as expected, so it's best not to run the gel for too long so the bands don't spread out too much (1 hour, 10% PA gel, 130V; with these conditions they are largely concentrated together). My Cas9 mRNA-only control was clean with no additional bands. I find this more sensitive than the surveyor assay, since you can load a lot of PCR product on the gel (i.e. more than the 400ng of Surveyor digested DNA), and there is no smearing to contend with.

Thanks for the tip Fatwa!

[Fatwa Adikusuma]

No worries. I found that technique from these papers 

http://www.ncbi.nlm.nih.gov/pubmed/23075543 

http://www.ncbi.nlm.nih.gov/pubmed/23573916

Cheers,

Fatwa

[Ram Ajore]

Hi fatwa,

Thanks for great idea....but i would like know still two PCR needs to be done separtely. one for wild type and one for mutant one and then hybridize them at 1:1 ration and run on acrylamide gel..

ram

On Wednesday, September 10, 2014 5:04:12 AM UTC+2, Fatwa Adikusuma wrote:

[JP]

Would you be so kind as to attach a photo, please?

[thomas cunningham]

Sure...

[JP]

Thanks!

[Fatwa Adikusuma]

I don't know what your case is, of course you don't need to do that when your samples are pool of cells. But we do that when we genotype homozygous mutant mice. Or you can also mix WT and mutant DNA prior to PCR.

Cheers,

Fatwa

[Ram Ajore]

Thanks fatwa...i got it now..

ram

[mario amendola]

Hi all,
To test designed CRISPR guide efficiencies, we recently developed in the lab an easy and fast method.
The method requires only a pair of PCR reactions of your CRISPR treated sample and a control sample, followed by two standard capillary sequencing runs (sanger sequencing). The sequence traces can then be uploaded on the following website: http://tide.nki.nl/

Read the details here: http://nar.oxfordjournals.org/content/early/2014/10/08/nar.gku936.abstract

Check it out, you will love it
Cheers
Mario

[Phil Abbosh]

Thomas and Fatwa
I tried the heteroduplex mobility shift assay and got some unexpected results:

lanes 1-4 were transfeced with sgRNA plasmid (pX330)
lane 5 untransfected parental cells
lane 6 BAC containing entire gene
lane 7 lane 5+lane 6
lane 8 positive controls C and G from surveyor kit

my PCR product is 1kb.  i dont understand why i am getting so many bands in lanes 5 and 6.  I thought there might be contamination with a template containing SNPs or some other mutant but there is not (i checked).  There is only one copy of the tumor suppressor gene i am working on (the other is lost in this cancer cell line).  i also thought it might be from an error-prone taq so i tried a proofreading enzyme (platinum Pfx; i was using platinum taq)-same results.  anyone seen anything like this or have an idea of how to clean it up? 

[thomas cunningham]

Yeah, I don't think those bands are heteroduplexes -- although lane 7 looks like there is. How do you do your heteroduplex formation? Have you tried a temperature gradient to optimize the PCR? I use a hot start proofreading taq (Phusion flex) -- perhaps that reduces non-specific bands?  I recently ran out, and had to use an expired (non-hot start) Phusion, and I got multiple non-specific bands above and below my specific band. I haven't tried G and C controls with this method yet.

[Phil Abbosh]

thanks for responding Thomas.  definitely think there are heteroduplexes in lane 7 (although i would expect only one band; there is a nonsense mutation in this cell line).  i did a Tm gradient for my conditions and optimized [Mg] and [primers] too.  maybe i should try a different brand of Taq. I sent the products off for sequencing to try TIDE that is mentioned in all the recent threads as well. 

[Varsha Bhargava]

Hey Phil,

   My PCR product is the same size as yours and I ran it on a 10%gel, which I am guessing was a bad idea. What percent PAGE did you run?

Thanks!

Varsha

Regards,

Varsha Bhargava

Graduate Student

Buszczak Lab

Graduate School of Biomedical Sciences

University of Texas Southwestern Medical Center at Dallas

On Thu, Oct 16, 2014 at 5:18 PM, Phil Abbosh <jager...@gmail.com> wrote:

thanks for responding Thomas.  definitely think there are heteroduplexes in lane 7 (although i would expect only one band; there is a nonsense mutation in this cell line).  i did a Tm gradient for my conditions and optimized [Mg] and [primers] too.  maybe i should try a different brand of Taq. I sent the products off for sequencing to try TIDE that is mentioned in all the recent threads as well. 

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[Phil Abbosh]

Varsha
That PAGE was a 4-20% gradient that the Zhang lab and others have recommended. Did you get a similar banding pattern?

I have since moved to 4% and that also showed the same banding pattern.  I have actually narrowed the problem down to something in the PCR: that banding pattern occurs even if i dont heteroduplex. The un-heteroduplexed products are the appropriate length on an agarose gel, so it never occurred to me to run un-heteroduplexed PCR product on a PAGE, but when i do, i get innumerable bands above the right-sized band even from template from cells which were not transfected and from BAC template.  I tried 3 taq's and 2 different primers that reliably give one crisp band on agarose gels and still always get innumerable bands.  i am re-optimizing the reaction conditions and the reaction is getting cleaner.  i can keep you updated if you like.

[NMR]

Thanks Mario, 

Do you think TIDE would also work with clonal lines? We are interested in biallelic disruption by NHEJ indels. We mostly find that the indels found in both alleles differ from each other. This make it really difficult to resolve the complex double-peak-containing chromatograms. I am wondering if there's any reason why TIDE would not work for clones?

Many thanks and great job, 

[Duško Lainšček]

in need of help;

I am trying to astablish T7 endonuclease assay in our lab, but i have problems.I also got double bands in my wt,as it in wt there is a heteroduplex formation.I know about PAGE,but i need to have cut bands and uncuts,because i need to know about % of HDR and NHEJ,

Does anyone have a solution for me?

I would be very helpfull, because it is getting very frustrating..

thx

best

Dusko

--

[mario]

Hello,
I'm not sure I completely understand your question. Tide will tell you if the two sequencing reaction are different (in which way and in which percentage), whatever is your starting material.
However, since the procedure is very simple and fast, I always suggest to just try it
Let me know if you need any further clarification
Cheers
Mario

[Fatwa Adikusuma]

I would say, try the PA gel first to make sure you get heteroduplex. When you know you get heteroduplex, you can continue the T7 assay. However, T7 assay or Surveyor assay is not very accurate predicting the efficiency. RFLP is more accurate, and if you don't have a restriction site, you can do Cas9 RFLP. Here is the paper about that http://www.ncbi.nlm.nih.gov/pubmed/24445736 .  You can find the protocol for Cas9 RFLP in NEB website.

If you share your T7 protocol, people here might be able to correct if there is a mistake.

Cheers,

Fatwa

[Fatwa Adikusuma]

Yes I think it is easier to use TIDE to quantify the efficiency in pool of cells. You must try it too.

Cheers,

Fatwa

[Jivan Khlghatyan]

Hello.

I tried TIDE too and it works well. But the results differ from what I got with SURVEYOR assay. While with surveyor I was getting 8% TIDE showed me 17%. Does anyone observe similar things?

Thanks

[Razieh Monjezi]

Hi Jivan,

could you please let me how you did the TIDE, I mean regarding the sample preparation, we should amplify the taregt region with the primers that we use for SURVEYOR or we should have a larger PCR product using different primers, since for my SURVEYOR assay my PCR product size is 500-600 bp, and for sequencing can we use the same primers as SURVEYOR, we should use both forward and reverse primer or one should be enough?

Thank you for your consideration.

regards

Razieh

[Harrys Kishore Charles Jacob]

Hi Jivan,

We have had similar results as well where TIDE seems to overpredict the indel frequency. T7endonuclase assays shows something in the range of 15% while TIDE around 62%. Do others see similar results as well.

CJ

On Thursday, November 13, 2014 at 9:30:04 PM UTC+1, Jivan Khlghatyan wrote:

[Fatwa Adikusuma]

Hi CJ, TIDE works well to me, and consistent with analysis using RFLP assay. I am wondering If you do independent analysis (TIDE and T7E), are you going to get replicable results from what you previously found?

Cheers,

Fatwa

[Marta Coll]

Hello everyone,

 

I also was having troubles with the surveyor assay, so once I found this conversation I quickly tried the gel electrophoresis and it worked! Now the trouble comes in analyzing the bands, I am a bit confused with it, so if anyone could help me I would be very grateful.

 

In my case I wanted to check the DSB efficiency that different RNA guides where leading to, so that I could choose the RNA guide leading to the best DSB efficiency to continue my experiments. I tested 3 different guides for each two different exons.

 

For it, I transfected 293T cells with my plasmid that contains the RNA guide and the nuclease. The transfection efficiency was 80%. Then I extracted the DNA of the cells and performed a PCR of the region surounding the place where the DSB should occur (550 bp). I re-annealed the DNA strands and performed the gel electrophoresis.

 

So theoretically I had for each of the guides I tested:

20% Untransfected cells --> wt DNA

80% Transfected cells --> random pool of indels that happened in each cell

 

Now considering this I would be expecting to have a thick band of homoduplex, and above it many multiple heteroduplex bands representing different indels that happened randomly in different cells. This happened in the guides 1 and 2 (exon 6) and guide 5 (exon 5).

However in the guide 13 (exon 6) just one heteroduplex bands can be seen, and I don't know if I am interpreting the result correctly. Does this mean that just one single event of repair happened in one cell, and the bands I can see is from that cell and the clonal ones that grew from that one? And would this mean my guide 13 (exon 6) had a worse DSB efficiency than the others?

 

Another question is, in the control from the Exon 5 (C), I can see two other bands, but that pool of cells was not transfected with any RNA guide nor nuclease. What could those bands mean since those bands are present exactly the same way in the guide 13 (exon 5)?

 

Thank you in advance for the help!

 

Marta

[Fatwa Adikusuma]

Hi Marta, your guides seem working except for g13 exon 5. If you have a look that for exon 5 the WT band give extra bands which also present in other samples, that means those extra bands are background. I really suggest to use TIDE analysis to analyse the percentage since I found it is very reliable. Just make sure you have a good sequencing chromatogram, I found cleaning up the BDT reaction using magnetic bead based method works beautifully.

Cheers,

Fatwa

[Marta Coll]

Thanks for your opinion Fatwa! I will go for the sequencing as well to see what I get. Thank you very much for your suggestions and good luck with your own work.

Cheers, 

Marta

El divendres, 6 maig de 2016 4:36:38 UTC+2, Fatwa Adikusuma va escriure: