We also digest the plasmid in our supes after transfection for lentivirus production because we don't want to detect the plasmid rather than integrated virus. Our protocol is to digest with crude DNas I (rather than qPCR quality, we purchase from Calbiochem cat #260913), at least 10 U per ug plasmid DNA used to transfect. We supplement the supe with 10 mM MgCl2 (I make a 2 M MgCl2 stock in water, sterile filter) at the time of digestion. The DNase I unit activity varies from lot to lot, but we dilute it down to 30,000 U/ml (=30 U/ul). Since I typically transfect with 30 ug DNA in 25 ml media, I add 10 ul DNase I along with 125 ul of MgCl2. I digest at room temperature for 60 min but some people in lab go for 30 min at 37C - either way is fine. We also typically concentrate our virus preps by ultracentrifugation on a 5% sucrose cushion at 25-30K rpm, which helps eliminate the excess calcium for downstream applications since some cells are calcium sensitive. Our lab has done extensive analysis on these virus preps/infected cells and find this protocol eliminates plasmid DNA and downstream qPCR/qRT-PCR assays fail to detect virus in supe only.

Try adding some Restriction enzyme the Mix...

Cut the PBS into smaller pieces using enzyme that is not in the insert.

Thanks Priscilla

Prashanth:I cant understnad what point you are making with RE mix?

Shi-Hua:I am not cloning something, I want digest the plasmid DNA so as it wont be there active for my subsequent reaction

If I am not wrong, u want to transfect viral cDNA and analyse by qRT PCR. But when you Isolate RNA ( due to the buyout density of plasmid) you get plasmid in your RNA. right??

Dnase I is very effective in digesting linear DNA and I am not sure about its activity to digest plasmid DNA.

i)Try Isolating RNA with LiCl( as it selectively precipitates RNA)

ii)Try designing primer within intron exon junction of gene if your viral cDNA has one of them.

iii) add RE+ Dnase I before u make cDNA.

Hope It was helpful.

Thanks for your comments.

no, I am not interested with any RNA either, I want to transfect viral vectors to make virus, and after transfection we can collect them in the supernatant,you know. There is a possibility that, any remnant plasmid DNA cound be found in the supernatant as well(very minute). This contamination subsequently reflect in my infection and the qPCR I make with the infected cell genomic DNA(integrated viral DNA in cell line is all I need, not the one in the plasmid!).

Check the promega's homepage. DNase I requires Mg++/Ca++ and when you add Mn, DNase I become able to cause double-strand breaks if my memory serves correctly. You do not need to use buffers commercially attached, but you need Mg++ at least in your solution to digest DNA.

Yes Mr.Kazuo, I was making a research in all these company websites and I got this idea! thank you very much for your advice anyways. :)

We also digest the plasmid in our supes after transfection for lentivirus production because we don't want to detect the plasmid rather than integrated virus. Our protocol is to digest with crude DNas I (rather than qPCR quality, we purchase from Calbiochem cat #260913), at least 10 U per ug plasmid DNA used to transfect. We supplement the supe with 10 mM MgCl2 (I make a 2 M MgCl2 stock in water, sterile filter) at the time of digestion. The DNase I unit activity varies from lot to lot, but we dilute it down to 30,000 U/ml (=30 U/ul). Since I typically transfect with 30 ug DNA in 25 ml media, I add 10 ul DNase I along with 125 ul of MgCl2. I digest at room temperature for 60 min but some people in lab go for 30 min at 37C - either way is fine. We also typically concentrate our virus preps by ultracentrifugation on a 5% sucrose cushion at 25-30K rpm, which helps eliminate the excess calcium for downstream applications since some cells are calcium sensitive. Our lab has done extensive analysis on these virus preps/infected cells and find this protocol eliminates plasmid DNA and downstream qPCR/qRT-PCR assays fail to detect virus in supe only.

I agree with Emily Lowe's suggestion.Hope this might have soled your problem. Wish you good luck. A.M.Jana

I think I should add this one. I remember that phosphate ion can inhibit or repress DNase I activity. You might want to avoid phosphate in your reaction mixture in some way.

With regard to Prashanth'sinitial comment. You could use RE such as DpnI which cuts GAmTC sites - that is only in DNA produced by bacterial cells. This process is used in site directed mutagenesis studies to remove the orginal template. As the methylation pattern is specific to bacteria DpnI will not cut PCR products or DNA from eurkaryotic cells. However given the volumes involved in treating supernatant & that your viral genome is protected by the virus structure I doubt an RE approach is worth while or likely to work given that an RE would most likely be more sensitive to reaction conditions compared to DNAse. There is generally no substitute for experience so I'd be going with Emily's excellent & detailed suggestion.

Yes I do follow the same idea give by Emily. Thank you all for you valuable comments! hope it will work this time :)

Emily Lowe & Ananda Ayyappan J.V. I m doing transfection too, in HEK293 viral packaging cell line, but didnt use DNase1.Collect the supernatant and than infect the cells. But only problem i have..In detect GFP expression in few cell, but the desire gene which i want to express is good. Can you kindly help me why GFP express in few cells?

Muhammad - Ah, we have ALL had THAT problem at one time or another! First, I don't use a packaging cell line. Instead, I use the various packaging plasmids (Gag-Pol-Rev, VSV-G, lentiviral vector) in transfection. Regardless, maybe I can help...I have several questions:

1) Do you titer and if so, on what cell type and what are the titers (based on either GFP or your desired gene or both)? Depending on the size of my constructs, I typically get 5x10^4-5x10^6 IU/ml in 293T.

2) If you are using a lentiviral system, are your p24 values high (a good virus prep for me would be >2000 ng/ml)?

3) How are you driving GFP? IRES, 2A, or a separate promoter (and which one, if so)?

4) What is the order of your vector (Desired Gene-GFP or GFP-Desired Gene)?

5) Do you have an "empty vector" control with just GFP (cutting our your desired even)?

6) In which cell types is GFP not expressing well?

7) Simple, I know, but have you sequenced and digested your plasmids to be sure that GFP is in frame and your plasmids are a single, clean species? I ask because lentiviral vectors recombine so easily (due to the LTRs) and strict single colony amplification is necessary to prevent mixed species in your plasmid prep.

I typically have problems seeing high GFP in certain hematopoietic T cell lines (like CEM.NKR, CEM.T1, CEM.T2, MOLT4, Jurkats), and primary T cells and macrophages when GFP is after a large gene despite excellent GFP expression in 293T cells. So the size of the gene upstream of GFP is important. The promoter driving the gene is important (I have become quite fond of SFFV for strong promotion). I also switched to ZsGreen (Clontech), which is several logs brighter than GFP and EGFP.

Thank,s for your reply in detail. I am using plasmid for transfection in HEK293..Than infect by recombinant adenovirus bone marrow mesenchymal stem cells.

Before transfection for confirmation i run the plasmid on agarose gel..digested and nondigested plasmid.

Not titer the virus yet..but my supervisor suggested to me,, do viral titer i will do that.

GFP not express well in Mesenchymal stem cell.

Please find further detail of the plasmid construct http://www.addgene.org/19412/.

Thank,s again. let me know what i should have to do..to get good GFP expression.

Alas, I do not work with adenovirus so I'm not sure I can be of much help. But I would think the basics are the same.

Upon looking at the construct in Addgene, it looks like the gene is driven by a CMV promoter and then an IRES to the GFP. I do know some IRES sequences simply don't express well in some cell types...You might try expressing your vector in different cells types to see if this is a phenomenon in only of your mesenchymal cells, in which case changing the IRES to a 2A sequence or even another promoter entirely might help. Another option is to replace GFP for something like LNGFR, which sometimes expresses better. It could be the cell type or the sequence or the promoter, who knows, but we improved our marker expression when we switched out GFP for LNGFR in one of our lento-vectors.

Also, I'd double check your sequence. You must not have a stop codon after your desired gene before the IRES. A stop codon will prevent GFP from being made. I strongly suggest you sequence your plasmid.

You need to figure out a way to titer your virus. I had a very complicated lentiviral vector that contained an shRNA, a 1500 bp gene and then ZsGreen. The ZsGreen (the third gene in the construct) was being silenced for unknown reasons and there was no antibody to the 1500 bp gene of interest, so I developed a cell line that overexpressed the gene the shRNA targeted and calculated a relative titer based off of the shRNA knockdown. You just might have to get creative to measure titer - too much virus can cause cells to shut down protein production, so knowing how much virus you are giving them is important.

Alternatively, you can determine how important that marker gene (GFP) really is. If it's not really that helpful anyway, remove is entirely and focus on a way to see your desired gene. A biological assay perhaps if your gene leads to secretion, I don't know.

I'm not sure I was even of any help but I hope you get things working soon!

Many many thank's. Thats so much infomation regarding my GFP problem. I will infect diffrent type cell to see how GFP express.

Hi Ananda Hope u are doing fine.

I have one more suggestion to give to you.

This is a very widely used technique, mostly by plant virologist.

Its called as IC RT PCR. Its a combination of ELISA and RT PCR. Hopefully, if u have any antibodies against ur viral particle, you can use in this technique.

Secondly u can always run a RT - PCR and subtract the difference in ct from the test.

Hope it Helps.

Thank you.

Belated Happy Diwali

Convey my regards to Sathish.

Hey Prashanth and All,

I could successfully make it with the fermentas Dnase I 10X reaction buffer(containing MgCl2)+additional Cacl2(1mM), and my PCR worked fine as we expected!

Happy Diwali to all and thank you very much,

Cheers,

Anand

Hi Eszter, So far I dont know anyone who deactivates DNAse I enzyme after digestion, since the intention is to kill the plasmid DNA, before the infection with viral particles. So viral (only) particles are going to infect the cells (attached/in suspension in the medium), hence I dont see a need for this! (If you think this protien hinders the virus or cells, remember the medium contains 10% FBS which is full of proteins!). So just go ahead!

Good luck!