Відео

excellent question

I am not sure. Transformation methods can be pretty specific.

Thank you for your encouragement!

Our pleasure!

Please go to my website and download a PDF protocol that has that information. Link is in the description.

This video is about how plants shed leaves. It is not clear what you are referring to.

Gracias

You are welcome

Please download the protocol which has the thermocycler program at: patharkar.com/2022/03/designing-and-making-crispr-cas9-plasmids-quick-easy

It helps the bacterial suspension better enter the flower and get to the ovule be breaking the surface tension of water. Silwet L-77 is a nonionic detergent.

Yes you can. Just a few seconds in liquid nitrogen and it will freeze. I am not sure about the DNA recovery between the freezing methods. The ice crystals will be different between a freezer and liquid N2.

@@RahulPatharkar Awesome! Also, What kind of recovery efficiency can you get with this method? Have you measured DNA quality (260/230) aswell? Im curious because i often get very poor 230 ratios when using comercial kits and I imagine it might be the reason why some of my transformations aren't working.

@@xdacunha Recovery varies between 50-90% related to how you cut out your gel and the size DNA. DNA from freeze and squeeze prep has ethidium with it that distorts the wavelength ratios, however, the DNA can be used in most downstream applications directly. I sometimes further purify the DNA with penol:chloroform:isoamyl alcohol which leaves exquisitely pure DNA.

Actually the technique is quite similar.

Thank you and good luck.

We appreciate your support!

You can do further cleaning if you like. However, I have done sequencing, cloning, digests,... with this DNA. it is pretty clean and by the way many of your points are actually in the the protocol on patharkar.com.

It has lasted 3 years so far. Held up pretty much perfectly. Some areas where I added the material a little thick did crack a little. Those minor crack took about 5 minutes to repair.

Different filter material is possible, however, I have used a number of brands successfully. One thing I noticed is that sometimes people do no realize your DNA can be quite dilute if a lot of extra non-DNA containing agarose is surrounds the DNA band. I always preferred to cut so that my eluted DNA would be as concentrated as possible.

@@RahulPatharkar thank you for your quick answer. We don´t have success with DNA gel extraction even with columns kits... We placed micrograms of DNA in the wells and obtained a few nanograms 5 ng/ul with the columns method.

Yes, but a regular freezer will suffice.

To get DNA or RNA out of a PAGE gel typically will require electroelution not a freeze and squeeze.

@@RahulPatharkar By 'electroelution on a free and squeeze' you mean instead of centrifugation to apply electroelution on the cutout, frozen fragment, or I undesrtood wrong? Also is the same method that you describe in the video good for RNA purification?

@@ChristinaPatra-il1jl Yes, electricity to get the nucleic acid out of the gel.

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A carefully taken image from your smartphone can work well.

Sorry, I do not have much experience with gram positive bacteria DNA extraction. Not sure if alkaline lysis works for gram positive. However, this method would likely cleanup DNA from lysed bacteria.

Arabidopsis is one of the easiest to transform. I have transformed soybean and pennycress as well. They were doable, just takes a little more effort. Other friends appear to make corn transformation look pretty easy as well.

Unfortunately, the lab equipment makes a lot of background noise that cannot be turned off.

​@@RahulPatharkar A clip-on microphone positioned closer to the mouth should pick up more voice and less noise. Still, this was interesting to watch.

The are welcome!

You are welcome!

Your are welcome.

You are most welcome

Unfortunately it is filmed in a lab with a fume hood and other equipment that makes noise. How would you suggest to handle this background noise?

PCI can be helpful for any DNA cleanup. I cannot recommend a lysis buffer for leeches because I never worked with them. You could try the buffer in this paper: jacks-lab.mit.edu/protocols/dna_isolation_proteinase_k_method

Your welcome.

Thank you

Not sure what exactly you mean but probably yes.

That is based on my data. The main benefit comes from having much less waste. You can try other ratios. For me all ratios I tested resulted in excellent sequence data. Importantly, almost everything extracted by PCI ends up in the organic:aqueous interphase and a small amount of PCI has a similar surface area for the interphase. Hope this helps.

@@RahulPatharkar Thank you. That's really helpful

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

Yes, any DNA can work.

Please check patharkar.com

Thanks! I hope it helps you.

Yes. You could use a kit or you could use the PCI method on this channel. Either will work to cleanup for sequencing.

Glad it was useful to you!

SnapGene

The segregation ratio is the simplest way to figure out zygosity. There are also PCR based methods but you typically need to know more about the molecular nature of your transgenic events.

All you can do now is put your DNA in the fridge for a day and vortex it some tomorrow and see what you can dissolve. Just keep the supernatant after vortexing. The DNA should dissolve if you give it time.

Thanks!

Observing the OD is not necessary. My overnight cultures are passed log phase. A wide range of ODs can yield transgenic plants. The stage of the plant is quite critical.

Golden Gate cloning which is shown in this video does insert a gRNA fragment into restriction sites. Please check the video carefully to see the restriction sites.

It would be best to growth known WT plants without selection. If possible also grow empty vector transgenic plants as a non-transgene control so that you have a control that can be treated with selection.

The video was done in Excel not Word.

At the beginning of the video, there is a word file displayed. This is what I mean..Can I get it?

@@solakhalid8967 That is made long time ago on a different computer. It would take a while to dig that up. Sorry.

@@RahulPatharkar thank you very much...The video helped me and I needed it

You just need to incorporate a repair template.

I just tried to download the Excel file protocol and it worked fine. Please try again or tell me what error you are getting and maybe I can help.

@@RahulPatharkar Yes you were right, thank you for the quick answer!