Hi ... Its a flowless wonderful video.... But my practical was not gone like this ...as i seen in the video activation buffer was spread in the leaf ...but when i was performed it was stuck ...it was not spread .... why it happened...could you please reply
One possible reason is that the stomata may not be open. Make sure to use well-watered plants earlier in the photoperiod. Plants close their stomata before the night cycle starts.
Plants pots are maintained in the culture room, where conditions regarding photoperiod are already pre_set...should I perform an experiment around 9-10 AM in the morning?
@@muktaprajapati2652 early to mid day have the stomata open the most. Your idea for 9-10am is good if that falls within the first half of the photoperiod.
Hi! My class linked to your video for one of our labs in Lab Techniques in Plant Studies, and I really enjoyed it and learned quite a bit. I work in accessibility, and had a mild concern about the music in the background. I was wondering if you wouldn't mind posting a transcript somewhere, or I could provide you one? Someone who has low hearing might have trouble understanding the video.
Glad it was helpful to you. The video has closed captions, you just have to click the "CC" button on UA-cam. Is that what you want? If you want just a text document you could make one and I can post it to the website?
Hi, Thanks for the video. Can you suggest to me how to prepare the sample for imaging purposes? Should I cut the infiltrated region into pieces and go for imaging directly or do I have to remove the epidermis layer?
You do not have to remove the epidermis, in fact the epidermal cells express and are probably what you will image. I suggest putting pieces of the leaf under a coverslip in a cavity slide (so that the leave does not get crushed) and then imaging.
Hi! Thanks for the video. I was wondering if you have information on what the vector design looks like? I understand you need a promoter for your gene of interest, usually a CaMV 35S promoter. However, would it be sufficient to only have the CAMV35S promoter and your gene of interest in the T-DNA region of agrobacterium to induce transient gene expression? Or are more things required in the T-DNA region?
please can you give me a detailed explanation/video on the following: 1.How to perform yeast two hybrid assay ;from the beginning till the end 2. How to perform obtain over expressing and CRISPR mutant lines of a gene of interest. Am working on tobacco. I really need this urgently. please help
Namaste, Rahul I'm very much interested in biopharmaceutical and wants to open my own green house in Assam or in Kashmir .. I think you would be of great help in future. Shukriya
Thanks for your reply Mr. Patharkar I have a question as a layman . I would like to ask you that in Himalayan region a particular flower is grown and that flower is good for heart ailments . So homopathic mother tincture is made to extract plant extract in ethonol. I would like to know can we take the cells from the flower and insert it on tobacco leaves and then take vaccine out from it .. Is this possible in leaf expression system or some other way. See there is a shortage of the flower as it grows only once in a year.
@@RahulPatharkar yes i read that in your protocol, Thank you. But do you have the Paper/book that mentions how exactly does it improve gene expression?
@@ahmedlabidi6795 It does it by suppressing silencing. Here is a paper: Lombardi, R., Circelli, P., Villani, M.E. et al. High-level HIV-1 Nef transient expression in Nicotiana benthamiana using the P19 gene silencing suppressor protein of Artichoke Mottled Crinckle Virus. BMC Biotechnol 9, 96 (2009). doi.org/10.1186/1472-6750-9-96
@@LungeloSikhakhane-x4e I do not have a video specifically for gene silencing. However, this agro method can be used to do it if you have the correct vectors.
Why you stopped making experimental vudeos? please continue. It really helps.
Thank you for your encouragement!
Thank you Catherine
You are welcome.
Informative video... 👍😊
👍
Hi ...
Its a flowless wonderful video....
But my practical was not gone like this ...as i seen in the video activation buffer was spread in the leaf ...but when i was performed it was stuck ...it was not spread .... why it happened...could you please reply
One possible reason is that the stomata may not be open. Make sure to use well-watered plants earlier in the photoperiod. Plants close their stomata before the night cycle starts.
Plants pots are maintained in the culture room, where conditions regarding photoperiod are already pre_set...should I perform an experiment around 9-10 AM in the morning?
@@muktaprajapati2652 early to mid day have the stomata open the most. Your idea for 9-10am is good if that falls within the first half of the photoperiod.
Thank you I wil definitely try next week
Verry much helpful to me. Please make a vedio about agro infiltration in onion plant
Actually the technique is quite similar.
Hi! My class linked to your video for one of our labs in Lab Techniques in Plant Studies, and I really enjoyed it and learned quite a bit. I work in accessibility, and had a mild concern about the music in the background. I was wondering if you wouldn't mind posting a transcript somewhere, or I could provide you one? Someone who has low hearing might have trouble understanding the video.
Glad it was helpful to you. The video has closed captions, you just have to click the "CC" button on UA-cam. Is that what you want? If you want just a text document you could make one and I can post it to the website?
Hi, Thanks for the video. Can you suggest to me how to prepare the sample for imaging purposes? Should I cut the infiltrated region into pieces and go for imaging directly or do I have to remove the epidermis layer?
You do not have to remove the epidermis, in fact the epidermal cells express and are probably what you will image. I suggest putting pieces of the leaf under a coverslip in a cavity slide (so that the leave does not get crushed) and then imaging.
Hi! Thanks for the video. I was wondering if you have information on what the vector design looks like? I understand you need a promoter for your gene of interest, usually a CaMV 35S promoter. However, would it be sufficient to only have the CAMV35S promoter and your gene of interest in the T-DNA region of agrobacterium to induce transient gene expression? Or are more things required in the T-DNA region?
Yes that would work for transient expression. Typically a tag would be added to the protein so that the protein could be visualized.
@@RahulPatharkar Thanks for your help!
A print protocol to go with the video can be downloaded here:
patharkar.com/2019/02/tobacco-agroinfiltration-transient-transformation
Thank you
please can you give me a detailed explanation/video on the following:
1.How to perform yeast two hybrid assay ;from the beginning till the end
2. How to perform obtain over expressing and CRISPR mutant lines of a gene of interest. Am working on tobacco.
I really need this urgently. please help
Thank you
Print protocols and more background can be found on patharkar.com
Namaste, Rahul I'm very much interested in biopharmaceutical and wants to open my own green house in Assam or in Kashmir .. I think you would be of great help in future. Shukriya
So nice of you
Thanks for your reply Mr. Patharkar I have a question as a layman . I would like to ask you that in Himalayan region a particular flower is grown and that flower is good for heart ailments . So homopathic mother tincture is made to extract plant extract in ethonol. I would like to know can we take the cells from the flower and insert it on tobacco leaves and then take vaccine out from it .. Is this possible in leaf expression system or some other way. See there is a shortage of the flower as it grows only once in a year.
Thank you for the video. It wasn't possible to download the protocol. Is it still available? Thank you
Please try the links now. I had to fix things because my free url only works for the first part of the month, then I exceed the allowed bandwidth.
Link still not working
may i ask, what did you use the P19 protein for? is any gene silencing happening?
p19 is a silencing suppressor. It allows your proteins of interest to be expressed at higher levels.
@@RahulPatharkar yes i read that in your protocol, Thank you.
But do you have the Paper/book that mentions how exactly does it improve gene expression?
@@ahmedlabidi6795 It does it by suppressing silencing. Here is a paper: Lombardi, R., Circelli, P., Villani, M.E. et al. High-level HIV-1 Nef transient expression in Nicotiana benthamiana using the P19 gene silencing suppressor protein of Artichoke Mottled Crinckle Virus. BMC Biotechnol 9, 96 (2009). doi.org/10.1186/1472-6750-9-96
@@RahulPatharkar Do you have a video or published paper with regards to gene silencing in a plant?, if you do may you point to the right direction
@@LungeloSikhakhane-x4e I do not have a video specifically for gene silencing. However, this agro method can be used to do it if you have the correct vectors.