I have tested different antibiotics, the constructs have ampicillin resistency.

Lai: here goes the commercial interest of any vendor. We may have to depend on them unless until we have our own designed vectors. However, I am interested to know any inputs in this regard. 

Thanks. That's what I think. They regard this as proprietary.

Are you sure?  That sounds bizarre & hard to believe.  Did you call customer support and ask them?  

If that's really true, just try a bunch of antibiotics you have on the shelf... Of course it's going to be one of those :)

Thanks John. I only have Ampicillin and Kanamycin in the lab. What else are common for plasmid amplification in E.Coil?

For other plasmid sources like Addgene and DNASU, they have all the plasmid info and one can easily amplif the plasmid. But I guess they are non-profit in nature, so it could be different.

Chloramphenicol and spectinomycin are also common, buut see the more comprehensive list here:

http://blog.addgene.org/plasmids-101-everything-you-need-to-know-about-antibiotic-resistance-genes

Thank you very much, Christian!

Dear kwok,

I am also looking for answers to the same question. Were you able to get some info?

thanks

Hi Ashutosh,

I have not yet tried different antibiotics for transformation to amplify the CRISPR plasmids. Indeed I am still waiting for the delivery of the HDR plasmids. I ordered them in Jan but it takes more than one month for the CRISPR KO plasmid to come, and the HDR plasmid still not yet arrive....Once I got the plasmids I will try in HEK-293T cells first to see how they work, and 20ug plasmids are enough for that purpose. If the plasmids work well then I will think about amplifying them for transfecting other cell types (may be just try a whole bunch of antibiotics). 

I have tested different antibiotics, the constructs have ampicillin resistency.

Hi Roman, thanks for your information! I will try it then!

Hi Kwok, I'm also very interested in purchasing Santa Cruz's CRISPR/CAS9 KO plasmids. Are you done with the verification of silencing efficacy? Is it good enough?

Hi Ting, unfortunately the HDR plasmids are still not ready from Santa Cruz. Have been keep waiting...

Hello! Thank you for the Ampicilin information.

However the tube sent by SC contains three different plasmids with three different RNA guides, if I'm not mistaken. In that case, do you just pull several colonies after plating on your LB agar plate?? Selecting just one colony might strongly decrease your recombination efficiency... 

Would you guys pull together several colonies?

Cheers,

Zahra

Hi Zahra,

The HDR plasmids have arrived, so I guess I will try to amplify them soon. I will pick individual colonies and prepare the plasmids/sequence them seperately. You can always pool the three plasmids together again later during transfection  

Thanks for the informations Kwon. Hope to here from the HDR plasmids too.

Thank you very much for your reply Kwok-on. Good luck with your experiments!

Z.

Hello Kwok-On. I just received the CRISPR/Cas9 double nickase plasmid from Santa Cruz and want to amplify it. Could you please let me know if there was a specific type of competent E.coli you used. Also, how did you select the colonies containing the plasmid of interest?

Hi, I am also interested to know their HDR plasmid resistance marker as well. Please let me know. Thanks

Hi Madhumita and Nhat, we have used Ampicillin for transformation of TOP10 cells for both the knockout and HDR plasmids.

I just got the HDR plasmids too and also would like to amplify them. Any info would be really appreciated (antibiotic resistance, MCS info, seq primers, etc). Thanks!

Hi Kwok-on, I would like to know if you successfully amplyfied both plasmids, and if so , could you spare any details for the best way to do it? Thanks.

Dear Kwok, are you able to deliver the plasmids efficiently in human iPS cells? I hardly see GFP expression in hiPS cells but in HEK cells is working fine. Control plasmid from Lonza pmax-GFP work fine in hiPS. Any suggestions?

Hi Ashutosh, we have not tried transfecting human iPSCs with the CRISPR plasmids yet, but we tried mock transfection by emGFP using the Life Technologies nucleofector and is working fine. There were about 30% transfection efficiency and the iPSCs survived well and could be differentiated into NPCs. One thing is that the GFP signal will lose rapidly, which I guess makes sense because the cells were dividing and we did not put antibiotic for selection. In your transfection did you also include the HDP plasmid? It has puromycin-resistance gene so I guess if your cells survive in puromycin after transfection (but not the vector-transfected control) then the transfection should be ok and the CRISPR may work.

Hi Ricardo, ampicillin (50ug/ml) works well for amplifying the plasmids in E.coli.

Hi Kwok,

I'm currently trying to make KO with Crispr/Cas9 and HDR plasmids from SantaCruz in 2 cell lines (HeLa and 293T), and it's very difficult to obtain KO homozygotous due to the multiploidy of these cell lines. Did you succeed to manage it?

SCBT told me to make successive transfections but even doing it I did not succeed.

Do you have some tip to help?

Thank you for your help

Hello everyone,

We are very much interested in buying the CRISPR/Cas9 plasmids from Santa Cruz but are in confused state as many things from Santa Cruz don't end up working. Has anyone been able to achieve good knockouts with these plasmid constructs?

Thank you very much.

Priya

Hi everyone!

SantaCruz's CRISPR/Cas9 KO plasmids contain GFP - has anyone tried selecting successful knockouts using FACS sorting? (i.e. as an alternative to additionally buying the HDR plasmid and selecting using puromycin)

Many thanks,

Sam

Hi Priya and Sam,

We have transfected 293T cells using the KO and HDR plasmids together. After splitting the cells and put under selection in puromycin, we obtained clones (which might be mixed as we did not do FACS). We picked 9 or 10 clones and performed WB for the lysate and found that two of them showed reduced expression of the target protein (one have ~50% reduction and the other ~70-80%) but it was not 100% knockout. We also used the GeneArt cleavage kit form Life Tech and perform PCR to confirm the disruption of genomic DNA at the target site, but again the non-cleaved PCR product  was also observed, indicating the presence of the undisrupted gene. So I guess the CRIPSR should work but it might need single cell sorting or screen more clones in order to obtain homozygous knockout cells.

Hi Kwok,

I was wondering that how can you pick 9 or 10 clones? Maybe you did limiting dilution. If so, it would be a quite long time to wait them grow and perform WB.

Thanks,

Bin

You either have a mixed population or a heterzygous clone. Make sure you pick single clones by serial dilution as Bin Liu mentioned. Sometimes even a second serial dilution is necessary to isolate a single cell colony.

Hey everyone!

We also bought the CRISPR/Cas 9 plasmids for CD33 KO (both KO plasmids and HDR plasmids) from Santa Cruz.

I tried to do some transfection with THP-1 & HL60 cell lines. Using fluorescence microscopy, I was quite sure to have succesfully transfected at least some cells. But FACS analysis disabused me. We couldn't find any transfected cells, so I have to try some different methods and repeat the experiments.

So I guess I will also try to amplify the plasmids.

Does anyone know the size of these plasmids? Just an approximate value.

I also heard about the Amp-resistance, but the manuals/protocols from Santa Cruz don't share much information unfortunately.

Any ideas about the sequencing primers?

Thanks a lot!

HDR plasmids might be difficult to identify - but crispr pick 20 colonies  and use u6 primer to sequence for your sgRNA . THP-1 if would go for amaxa 4D or Mirus TransIT-2020 transfection reagent.  Using high amounts of CRISPR plasmids only without HDR will already give u enough signal to sort of FACS. With iPS we got 5-10 % GFP cells.

All the best.

Thank you very much for your answer. I will sequencing my colonies.

For THP-1 transfection we used Amaxa Nucleofector II with Nucleofector Kit T (because we could borrow this kit from colleagues to do some first trials). We succeded with transfection of an in-house GFP-vector and with the comprised pmaxGFP vector.

Maybe the point is that for the CRIPSR/Cas 9 induced KO we need to insert both plasmids at the same time.

Anyway, I will do some new experiments with modified protocol and different Amaxa Kit.

Hi Veronika, you may use the human U6 promoter sequence as primer to sequence the CRISPR plasmid

Hi Kwok-on, thank you for you answer! Do you know if U6 promoter also works for the HDR Plasmid?

Hi everyone,

we also bought the CRIPR KO and HDR plasmids from Santa Cruz. The amplification and idenditification of the KO plasmids worked fine with amp and the U6 primer. The amplification of the HDR plasmids worked also using amp. Has anyone of you tried to identify the different HDR plasmids by sequencing?

Hey Sebastian,

have you contacted Santa Cruz? We asked them to send us the 3 different 20 nt - sequences of our KO-plasmids and they replied very fast.

Maybe they will also give you these sequences for the HDR-plasmids? So you can try to sequence your clones.

Hey Veronika,

thanks for your reply. I already designed a sequencing primer, binding to the puromycin resistance and sequenced somes clones. The sequences I got, included one of the loxP sites and the beginning of the 3`homology arm.  I will now sequence more clones to identify the three different plamsids.

Kwok-On Lai

Hi everyone

I readed all the comments in this post, but just to be sure:

its possible to amplyfy your CRISPR santa cruz plasmid in E. coli using ampicilin ???

(I knwow that this post is from 4 years ago, but if someone answerme i will be very grateful)

Hi Gabriel,

We have amplified the plasmids last month. Ampicillin works

Thanks Gulnihal Ozcan

But Santa Cruz use a pool of three plasmids, each one with a different sgRNA, so when you amplified the plasmids, do you only amplifed one of these three plasmid?

(My doubt is because i bought the cas9 nickase Santa Cruz, so i need to amplify both plasmid of the pool)

You are welcome Gabriel Rodriguez

We have picked >6 colonies and amplified the plasmids seperately. After isolation of plasmid DNAs we have pooled them. The ratios for concentrations may not be 1:1:1 since we don't know which colony has which plasmid but by pooling we are increasing the chances of transfecting all there gRNAs to our cells.

Thanks Gulnihal Ozcan I´m going to try :D

Hello! I have experienced some issues with my CRISPR-Cas9 KO transfection from Santa Cruz, it seems like a high percentage of my cells entered necrosis. I am using human fibroblast and transfecting them with Lipofectamine 3000. Has anyone done a successful transfection? How much DNA have you added (my control samples were fine, so I suspect that the problem might be in the amount of DNA, but I am happy to hear suggestions)